Characterization of the geographical origin of Pecorino Sardo cheese by casein stable isotope ((13)c/(12)c and (15)n/(14)n) ratios and free amino acid ratios

The stable isotope ratios ((13)C/(12)C and (15)N/(14)N) of casein measured by isotope ratio mass spectrometry (IRMS) and some free amino acid ratios (His/Pro, Ile/Pro, Met/Pro, and Thr/Pro) determined by HPLC in samples of ewes' milk cheese from Sardinia, Sicily, and Apulia were found to be parameters independent of ripening time. Multivariate data treatments performed by applying both unsupervised (principal component analysis and cluster analysis) and supervised [linear discriminant analysis (LDA)] methods revealed good discrimination possibilities for the cheeses according to place of origin. In this respect, particularly significant were the variables Ile/Pro, Thr/Pro, (13)C/(12)C, and (15) N/(14)N ratios on which basis 100% discrimination and classification of the samples by LDA was obtained.     

Regional origin assignment of red wines from Valencia (Spain) by (2)H NMR and (13)C IRMS stable isotope analysis of fermentative ethanol.

The use of the stable hydrogen and carbon isotope ratios of fermentative ethanol as suitable environmental fingerprints for the regional origin identification of red wines from Valencia (Spain) has been explored. Monovarietal Vitis vinifera L. cvs. Bobal, Tempranillo, and Monastrell wines have been investigated by (2)H NMR and (13)C IRMS for the natural ranges of site-specific (2)H/(1)H ratios and global delta(13)C values of ethanol over three vintage years. Statistically significant interregional and interannual (2)H and (13)C abundance differences have been noticed, which are interpreted in terms of environmental and ecophysiological factors of isotope content variation. Multivariate discriminant analysis is shown to provide a convenient means for integration of the classifying information, high discriminating abilities being demonstrated for the (2)H and (13)C fingerprints of ethanol. Reasonable differentiation results are achieved at a microregional scale in terms of geographic provenance and even grapevine genotypic features.     

Beef authentication and retrospective dietary verification using stable isotope ratio analysis of bovine muscle and tail hair

Stable isotope ratio analysis (SIRA) was used as an analytical tool to verify the preslaughter diet of beef cattle. Muscle and tail hair samples were collected from animals fed either pasture (P), a barley-based concentrate (C), silage followed by pasture (SiP), or silage followed by pasture with concentrate (SiPC) for 1 year (n = 25 animals per treatment). The (13)C/(12)C, (15)N/(14)N, (2)H/(1)H, and (34)S/(32)S isotope ratios in muscle clearly reflected those of the diets consumed by the animals. By applying a stepwise canonical discriminant analysis, a good discrimination of bovine meat according to dietary regimen was obtained. On the basis of the classification success rate, the (13)C/(12)C and (34)S/(32)S ratios in muscle were the best indicators for authentication of beef from animals consuming the different diets. Analysis of (13)C/(12)C and (15)N/(14)N in tail hair sections provided an archival record of changes to the diet of the cattle for periods of over 1 year preslaughter.     

Volatile components and key odorants of fennel (Foeniculum vulgare Mill.) and thyme (Thymus vulgaris L.) oil extracts obtained by simultaneous distillation-extraction and supercritical fluid extraction

Volatile oil extracts of fennel seeds (Foeniculum vulgare Mill.) and thyme leaves (Thymus vulgaris L.) were obtained by simultaneous distillation-extraction (SDE) and supercritical fluid extraction (SFE) and analyzed by gas chromatography-mass spectrometry (GC-MS). In general, fennel oil extracted by SDE and SFE showed similar compositions, with trans-anethole, estragole, and fenchone as the main components. In contrast, thymol and p-cymene, the most abundant compounds in thyme leaves, showed big differences, with generally higher amounts of monoterpenes obtained by SDE. However, in this case, the differences between the extracts were higher. Key odorants of fennel seeds determined by gas chromatography-olfactometry (GC-O) showed similar patterns when applying SDE and SFE. trans-Anethole (anise, licorice), estragole (anise, licorice, sweet), fenchone (mint, camphor, warm), and 1-octen-3-ol (mushroom) were the most intense odor compounds detected in fennel extracts. Thymol and carvacrol, with oregano, thyme, and spicy notes, were identified as key compounds contributing to the aroma of thyme leaves.     

Multielement isotope analysis of bovine muscle for determination of international geographical origin of meat

Multielemental (C, N, H, S) stable isotope ratio analysis was used as an analytical tool to verify the geographical origin of beef from several European and non-European countries. Beef samples were collected from nine different countries, and the (13)C/(12)C, (15)N/(14)N, (2)H/(1)H, and (34)S/(32)S ratios of defatted beef were measured using isotope ratio mass spectrometry (IRMS). There were highly significant differences in the mean isotopic values of the beef from different countries. The results of discriminant analysis showed that the four isotope ratios were significant for the discrimination of geographical origin and that 84.9% of the samples were correctly assigned to the country of origin (82.2% when cross-validated). Beef was also classified according to geographical origin when additional information on different feeding regimens used in Ireland was included, with 85.0% of the samples correctly allocated and 82.9% cross-validated using the isotopic signatures. All of the Irish beef samples verifiable as pasture-fed beef were correctly classified and then cross-validated.     

Flavor authenticity studies by (2)h/(1)h ratio determination using on-line gas chromatography pyrolysis isotope ratio mass spectrometry

Based on (2)H/(1)H ratio measurements of commercial synthetic and "natural" references, the recently developed on-line gas chromatography pyrolysis isotope ratio mass spectrometry (HRGC-P-IRMS) technique was used to determine the delta(2)H(SMOW) values of the flavor compounds decanal, linalool, and linalyl acetate, as well as those of E-2-hexenal and E-2-hexenol in foods and essential oils. In preceding model studies, the influence of sample preparation steps (simultaneous distillation extraction, SDE; solvent extraction, SE; liquid liquid extraction, LLE) on the delta(2)H values was found to be negligible. For decanal, the typical (2)H abundance, with higher content of (2)H for synthetic material (delta(2)H(SMOW) from -90 to -156 per thousand) and lower (2)H content for natural references (delta(2)H(SMOW) from -138 to -262 per thousand) was observed. Although the delta(2)H data recorded for linalool did not allow one to distinguish between synthetic (delta(2)H(SMOW) from -207 to -301 per thousand) and natural (delta(2)H(SMOW) from -234 to -333 per thousand) materials, the situation was somewhat more encouraging for linalyl acetate; delta(2)H(SMOW) values from -199 to -239 per thousand and from -213 to -333 per thousand were found for synthetic and natural samples, respectively. E-2-Hexenal and E-2-hexenol showed clear-cut origin-dependent differences in their (2)H/(1)H ratios; that is, delta(2)H(SMOW) values from -14 to -109 per thousand and from -263 to -415 per thousand as well as from -41 to -131 per thousand and from -238 to -348 per thousand were recorded for products from synthetic and natural origins, respectively.     

Chemical compositions and antibacterial effects of essential oils of Turkish oregano (Origanum minutiflorum), bay laurel (Laurus nobilis), Spanish lavender (Lavandula stoechas L.), and fennel (Foeniculum vulgare) on common foodborne pathogens

Chemical compositions and inhibitory effects of essential oils of Turkish oregano (Origanum minutiflorum O. Schwarz & P. H. Davis), bay laurel (Laurus nobilis L.), Spanish lavender (Lavandula stoechas subsp. stoechas L.), and fennel (Foeniculum vulgare Mill.) on Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus were determined. After the essential oils were applied on the foodborne pathogens at doses of 0 (control), 5, 10, 20, 30, 40, 50, and 80 microL/mL, the resultant numbers of cells surviving were counted. Results revealed that all essential oils exhibited a very strong antibacterial activity against the tested bacteria (P < 0.05). Gas chromatography-mass spectrophotometry analyses revealed that carvacrol (68.23%), 1,8-cineole (60.72%), fenchone (55.79%), and trans-anethole (85.63%) were the predominant constituents in Turkish oregano, bay laurel, Spanish lavender, and fennel essential oils, respectively.     

Detection of sugar syrups in apple juice by delta(2)H per thousand and delta(13)C per thousand analysis of hexamethylenetetramine prepared from fructose

An improved procedure for determining (13)C and (2)H isotope ratios, using gas chromatography-isotope ratio mass spectrometry (GC-IRMS), has been developed for identifying the addition of low cost commercial sugar syrups to apple juices and related products. Isotopic techniques are commonly used to identify the addition of low cost sugars to fruit juices and are difficult to circumvent as it is not economically viable to change the isotopic ratios of the sugars. The procedure utilizes the derivative hexamethylenetetramine, which is produced through chemical transformation of a sugar degradation product and provides position-specific (13)C and (2)H ratios that relate to the parent sugar molecule. The new procedure has advantages over methods using nitro-sugar derivatives in terms of analysis time and sensitivity. The differences between the delta(2)H per thousand and delta(13)C per thousand values of the 100 authentic apple juices and beet and cane commercial sugar syrups permit their addition to be reliably detected.     

Characterization of some Italian types of wild fennel (Foeniculum vulgare Mill.)

Wild samples of Foeniculum vulgare Mill. (fennel) were collected from thirteen Italian localities at different latitudes and grown in field trials to evaluate their morphological and agronomic characteristics and essential oil compositions. All the parameters were recorded at full bloom stage, and the essential oils (obtained by steam distillation) were characterized by gas chromatography (GC) and GC/mass spectrometry (GC/MS). The morphological characteristics showed positive relations among the number of umbels per plant, the plant weight, and the umbel weight percentage, and between these three parameters and the oil content. The essential oils evidenced five chemical groups characterized by (1) alpha-phellandrene, methyl chavicol, and trans-anethole; (2) alpha-pinene, limonene, and trans-anethole; (3) methyl chavicol and alpha-phellandrene; (4) methyl chavicol and alpha-pinene; and (5) alpha-phellandrene.     

Tracing the source of cooking oils with an integrated approach of using stable carbon isotope and Fatty Acid abundance

We report a new approach to identify swill-cooked oils that are recycled from tainted food and livestock waste from commercial vegetable and animal oils by means of carbon isotope values and relative abundance of fatty acids. We test this method using 40 cooking oil samples of different types with known sources. We found significant differences in both total organic carbon isotope as well as compound-specific isotope values and fatty acid C(14)/C(18) ratios between commercial vegetable oils refined from C(3) plants (from -35.7 to -27.0‰ and from 0 to 0.15) and animal oils (from -28.3 to -14.3‰ and from 0.1 to 0.6). Tested swill-cooked oils, which were generally refined by mixing with animal waste illegally, fall into a narrow δ(13)C/fatty acid ratio distribution: from -25.9 to -24.1‰ and from 0.1 to 0.2. Our data demonstrate that the index of a cross-plotting between fatty acid δ(13)C values and C(14)/C(18) ratios can be used to distinguish clean commercial cooking oils from illegal swill-cooked oils.     

Identification by HPLC-DAD and HPLC-MS analyses and quantification of constituents of fennel teas and decoctions

Qualitative and quantitative differences among the constituents in various fennel (Foeniculum vulgare Mill., family Apiaceae) teas prepared by classical infusion, microwave decoction, and dissolution are reported. Different commercial starting materials, such as fruit (unbroken and crushed), four herbal teas, and two instant herbal teas were evaluated. Chlorogenic acid (1), quercetin-3-O-beta-D-glucuronide (2), p-anisaldehyde (3), and trans-anethole (4) were identified by HPLC-DAD and HPLC-MS as constituents of fennel teas. No coumarins, which are characteristic constituents of plants of Apiaceae family, were found. Trans-anethole (4), the main constituent of the essential oil, was present in all teas. In addition p-anisaldehyde (3), a degradation product of trans-anethole, was also identified in all teas with the exception of two samples. Chlorogenic acid (1) and quercetin-3-O-beta-D-glucuronide (2) were also present in all teas. In addition, minor unidentified flavonol constituents were found in two teas. Quality, activity, and safety of the content of the investigated preparations are also discussed.     

Authenticity assessment of gamma- and delta-decalactone from prunus fruits by gas chromatography combustion/pyrolysis isotope ratio mass spectrometry (GC-C/P-IRMS)

Authenticity assessment of gamma-decalactone (1) and delta-decalactone (2) from peach (Prunus persica var. persica), apricot (Prunus armeniaca), and nectarine (Prunus persica var. nectarina) was performed using gas chromatography-isotope ratio mass spectrometry (GC-IRMS) in the combustion (C) and pyrolysis (P) mode. In addition, commercially available synthetic (nature-identical) 1 and 2 as well as biotechnologically produced samples (declared to be "natural") were characterized by their delta(2)H(V)(-)(SMOW) and delta(13)C(V)(-)(PDB) values. For the Prunus fruits under study, rather narrow ranges of delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data of 1, varying from - 34.6 per thousand to - 38.4 per thousand and -160 per thousand to -206 per thousand, respectively, were obtained. Synthetic references of 1 showed delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data ranging from -27.4 per thousand to -28.3 per thousand and -151 per thousand to -184 per thousand, respectively. Samples of 1 declared to be "natural" exhibited ranges from -28.1 per thousand to -29.2 per thousand and -192 per thousand to -286 per thousand for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively. For 2 from peach, apricot, and nectarine, delta(13)C(V)(-)(PDB) values ranging from -34.0 per thousand to -37.9 per thousand were determined; the delta(2)H(V)(-)(SMOW) values ranged from -171 per thousand to -228 per thousand. The delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data for synthetic 2 were -28.2 per thousand and -171 per thousand, respectively, that is, similar to those of 2 from "natural" origin, ranging from -27.7 per thousand to -30.1 per thousand and -185 per thousand to -230 per thousand for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively. GC-C/P-IRMS allowed clear-cut analytical differentiation of the synthetic and "ex-plant" origin of 1 and 2, whereas narrow ranges of delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data were found for samples of synthetic and "natural" origin.     

Isotope analyses of groundwaters of the north African plain

Isotope analyses of 36 groundwater samples were performed as part of an extensive hydrogeological study in Tripolitania, Libya. The stable isotope ratios ²H/¹H, ¹⁸O/¹⁶O, and ¹³C/¹²C, and the radioactive isotope ratios ³H/¹H and ¹⁴C/¹²C were measured which reflect mixing, vertical and horizontal flow as well as the individual recharge conditions of the various aquifers. The majority of water samples showed very low ¹⁴C as well as ³H concentrations corresponding to reliable conventional ¹⁴C ages exceeding 11,000 yrs. The results of the isotope analyses confirm, complete, and refine the hydrogeologically derived concepts of the study area.     

A multianalytical approach for determining the geographical origin of ginseng using strontium isotopes, multielements, and 1H NMR analysis

Asian ginseng (Panax ginseng C.A. Meyer) is widely used as an Oriental medicine in the East Asian regions, particularly Korea and China. In the study, the strontium isotope ratios ((87)Sr/(86)Sr), multielements, and metabolite profiles of 35 ginseng samples collected from Korea and China were examined in an attempt to develop a method to distinguish the origin of ginsengs from the two countries. A multivariate statistical approach was performed to analyze the multielements and the (1)H nuclear magnetic resonance (NMR) data. Results of a t-test for Mg, Fe, Al, and Sc showed significant variation between Korean and Chinese ginsengs, indicating potential tracers for discriminating them. Discriminating between the ginsengs from the two countries was generally successful when both the (87)Sr/(86)Sr ratios and rare earth element (REE) contents were used together. Moreover, principal component analysis (PCA) derived from the (1)H NMR data revealed a significant separation between the ginsengs originating from the two countries. The major metabolites responsible for differentiation were sugars such as glucose, xylose, and sucrose. The results suggest that this multiplatform approach offers a comprehensive method to distinguish the origin of ginsengs.     

Authentication of vegetable oils by bulk and molecular carbon isotope analyses with emphasis on olive oil and pumpkin seed oil

The authenticity of vegetable oils consumed in Slovenia and Croatia was investigated by carbon isotope analysis of the individual fatty acids by the use of gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS), and through carbon isotope analysis of the bulk oil. The fatty acids from samples of olive, pumpkin, sunflower, maize, rape, soybean, and sesame oils were separated by alkaline hydrolysis and derivatized to methyl esters for chemical characterization by capillary gas chromatography/mass spectrometry (GC/MS) prior to isotopic analysis. Enrichment in heavy carbon isotope ((13)C) of the bulk oil and of the individual fatty acids are related to (1) a thermally induced degradation during processing (deodorization, steam washing, or bleaching), (2) hydrolytic rancidity (lipolysis) and oxidative rancidity of the vegetable oils during storage, and (3) the potential blend with refined oil or other vegetable oils. The impurity or admixture of different oils may be assessed from the delta(13)C(16:0) vs. delta(13)C(18:1) covariations. The fatty acid compositions of Slovenian and Croatian olive oils are compared with those from the most important Mediterranean producer countries (Spain, Italy, Greece, and France).     

Application of stable carbon isotope analysis to the detection of testosterone administration to cattle

The use of anabolic substances is prohibited in food-producing animals throughout the European Union. No method is available to reliably detect the misuse of natural hormones in cattle. A method was developed to detect the abuse of testosterone in cattle fattening. Synthesized testosterone is rather depleted in the (13)C/(12)C ratio. Hence, the method is based on gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of urine. To select testosterone metabolites and endogenous reference compounds (ERC), the concentration of urinary steroids of cattle was investigated. Dehydroepiandrosterone and androst-5ene-3beta,17alpha-diol were chosen as ERCs to show endogenous (13)C/(12)C ratios. Etiocholanolone and 5alpha-androstane-3beta,17alpha-diol were chosen as the most important testosterone metabolites. Other metabolites known from literature like epitestosterone were less promising. In principle, GC/C/IRMS is a nonspecific method because finally carbon dioxide is analyzed. Therefore, a dedicated cleanup procedure for the selected steroids was developed. By means of proposed confidence intervals in the isotopic composition of ERCs and metabolites, the administration of testosterone to cattle could be detected reliably. Differences of up to 11 per thousand on the delta-scale between ERC and testosterone metabolites were found after testosterone administration, whereas endogenous differences did not exceed 2 per thousand.     

Oil stability prediction by high-resolution (13)C nuclear magnetic resonance spectroscopy

(13)C NMR spectra of oil fractions obtained chromatographically from 66 vegetable oils were obtained and analyzed to evaluate the potential use of those fractions in predicting oil stabilities and to compare those results with oil stability prediction by using chemical determinations. The oils included the following: virgin olive oils from different cultivars and regions of Europe and north Africa; "lampante" olive, refined olive, refined olive pomace, low-erucic rapeseed, high-oleic sunflower, corn, grapeseed, soybean, and sunflower oils. Oils were analyzed for fatty acid and triacylglycerol composition, as well as for phenol and tocopherol contents. By using stepwise linear regression analysis (SLRA), the chemical determinations and the (13)C NMR data that better explained the oil stability determined by the Rancimat were selected. These selected variables were related to both the susceptibility of the oil to be oxidized and the content of minor components that most contributed to oil stability. Because (13)C NMR considered many more variables than those determined by chemical analysis, the predicted stabilities calculated by using NMR data were always better than those obtained by using chemical determinations. All these results suggest that (13)C NMR may be a powerful tool to predict oil stabilities when applied to chromatographically enriched oil fractions.     

Detection of adulteration in Australian orange juices by stable carbon isotope ratio analysis (SCIRA)

Stable carbon isotope ratio analysis (SCIRA) was used to determine the authenticity of commercial Australian orange juices. Thirty-five samples of Valencia (delta(13)C values from -23.8 to -24.7 ppt) and eight samples of Navel juices (delta(13)C values from -24.1 to -24.5 ppt) of known origin were used to establish a decision level before analysis. No significant seasonal variations in (13)C/(12)C ratio were observed. Variations in combustion temperature in the method were also found to be insignificant.     

Determination of 2H/1H and 13C/12C isotope ratios of (E)-methyl cinnamate from different sources using isotope ratio mass spectrometry

For the authenticity assessment of (E)-methyl cinnamate from different origins, combustion/pyrolysis-isotope ratio mass spectrometry (C/P-IRMS) was used by an elemental analyzer (EA) and on-line capillary gas chromatography coupling (HRGC-C/P-IRMS). For that reason, (E)-methyl cinnamate self-prepared from synthetic, natural, and semisynthetic educts was analyzed in comparison to the commercial synthetic and natural ester. In addition, (E)-methyl cinnamate from basil extract and a number of commercial natural aromas was investigated. The data of self-synthesized synthetic (E)-methyl cinnamate, i.e., delta(13)C(V)(-)(PDB) = -33.8 per thousand and delta(2)H(V)(-)(SMOW) = +349 per thousand, corresponded with that found for the commercial synthetic samples (-29.5 to -31.4 per thousand and +328 to +360 per thousand for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively). The ester produced from natural educts by acid as well as Candida antarctica catalysis revealed delta(13)C(V)(-)(PDB) = -25.6 and -30.1 per thousand as well as delta(2)H(V)(-)(SMOW) = -162 and -169 per thousand, respectively. Acid-catalyzed semisynthetic products differed in their delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) values depending on the origin of their educts. For the ester from synthetic methanol and natural cinnamic acid, -27.3 and -126 per thousand were determined for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively, whereas for the ester produced from natural methanol and synthetic acid delta(13)C(V)(-)(PDB) = -30.6 per thousand and delta(2)H(V)(-)(SMOW) = +287 per thousand were found. Basil extract showed -28.9 and -133 per thousand for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively. Commercial aromas declared to be natural revealed delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data ranging from -25.7 to -28.5 per thousand as well as -85 to -191 per thousand, respectively, indicating, in part, incorrect declaration.     

Stable carbon isotopic composition of the wine and CO2 bubbles of sparkling wines: detecting C4 sugar additions

Sparkling wines have become a popular beverage in recent years, and the production of these wines is subject to adulteration during fermentation. This study investigated the stable carbon isotopic composition (expressed as delta(13)C) of the wine and of the CO(2) bubbles produced during the second fermentation for a number of sparkling wines produced in different countries around the world. Carbon isotope ratio analyses were used to estimate the addition of sugar obtained from C(4) plants (sugar cane or corn). The average delta(13)C values of the Brazilian brut, demi-sec, and doux sparkling wines were -20.5 +/- 1.2 per thousand (n = 18), -18.1 +/- 1.3 per thousand (n = 9), and -15.8 per thousand (n = 1), respectively. These values were statistically heavier (more positive carbon isotope ratio values) than the average delta(13)C of sparkling wines produced in other parts of South America (Argentina and Chile, -26.1 +/- 1.6 per thousand, n = 5) and Europe (France, Germany, Italy, Portugal, and Spain, -25.5 +/- 1.2 per thousand, n = 12), but not statistically different from sparkling wines produced in the United States or Australia. The most likely explanation for differences in the carbon isotope ratios of wines from these different regions is the addition of C(4) sugar during the production of some sparkling wines from Australia, Brazil, and the United States. The isotopic composition of the CO(2) bubbles (delta(13)C-CO(2)) followed similar trends. The average delta(13)C-CO(2) of most of the Brazilian and Argentine sparkling wines was -10.8 +/- 1.2 per thousand (n = 23), indicating that the likely source of carbon for the second fermentation was sugar cane. Conversely, the average delta(13)C-CO(2) of most of the sparkling wines produced in Chile and Europe was -22.0 +/- 1.2 per thousand (n = 13), suggesting that a different sugar (most likely sugar beet) was most used in the second fermentation. It was concluded that in many cases, the carbon isotope ratios of sparkling wine and CO(2) bubbles can provide valuable information about the sugar sources.