Luana Bontempo

Isotopic and Elemental Data for Tracing the Origin of European Olive Oils

H, C, and O stable isotope ratios and the elemental profile of 267 olive oils and 314 surface waters collected from 8 European sites are presented and discussed. The aim of the study was to investigate if olive oils produced in areas with different climatic and geological characteristics could be discriminated on the basis of isotopic and elemental data. The stable isotope ratios of H, C, and O of olive oils and the ratios of H and O of the relevant surface waters correlated to the climatic (mainly temperature) and geographical (mainly latitude and distance from the coast) characteristics of the provenance sites. It was possible to characterize the geological origin of the olive oils by using the content of 14 elements (Mg, K, Ca, V, Mn, Zn, Rb, Sr, Cs, La, Ce, Sm, Eu, U). By combining the 3 isotopic ratios with the 14 elements and applying a multivariate discriminant analysis, a good discrimination between olive oils from 8 European sites was achieved, with 95% of the samples correctly classified into the production site.

Influence of dietary composition on the carbon, nitrogen, oxygen and hydrogen stable isotope ratios of milk

The stable isotope ratios ((13)C/(12)C, (15)N/(14)N, (18)O/(16)O, D/H) of animal feed and milk were investigated, considering cows stabled in two farms and fed with diets made up of different kinds of C(3) plants and different amounts of maize. Maize was characterised by delta(13)C, delta(18)O and deltaD values significantly higher than those of the C(3) plants, while, for the C(3) plants, Festuca arudinacea had significantly higher content of (13)C and (15)N. The delta(13)C and delta(18)O values of the overall diet and the delta(13)C of milk casein and lipids were shown to be significantly correlated with the percentage of maize in the animal diet. On the other hand, the delta(18)O values of milk water and the delta(18)O, deltaD and delta(15)N values of casein were shown to be only slightly influenced by the amount of maize in the feed, being probably more closely correlated with the geo-climatic and pedological characteristics of the area of origin and with the presence of fresh plant or silage in the ration. The delta(13)C value of casein was shown to be a suitable parameter for evaluating the amount of maize in the diet: each 10% increase in the maize content corresponded to a shift of 0.7 per thousand to 1.0 per thousand in the delta(13)C of casein. A threshold value of -23.5 per thousand for delta(13)C in milk casein, above which it is not possible to exclude the presence of maize in the diet, was suggested. The results obtained could be useful for determining mislabelling of dairy products declared to have been produced by pastured animals or of PDO cheeses with an established amount of maize in the diet and for verifying the unpermitted addition of exogenous components to milk.

Multielement (H, C, N, O, S) stable isotope characteristics of lamb meat from different Italian regions

The study focuses on the (2)H/H, (13)C/(12)C, (15)N/(14)N, (18)O/(16)O and (34)S/(32)S values of defatted dry matter (DFDM) and on the (2)H/H, (13)C/(12)C and (18)O/(16)O values of the fat fraction of meat samples from various lamb types reared in seven Italian regions, following different feeding regimes (forage, concentrate, milk). The (13)C/(12)C (r = 0.922), (2)H/H (r = 0.577) and (18)O/(16)O (r = 0.449) values of fat and DFDM are significantly correlated, the fat values being significantly lower for C and H and higher for O than for DFDM values and the differences between the two fractions not being constant for different lamb types. The feeding regime significantly affected the (13)C/(12)C, (15)N/(14)N, (18)O/(16)O and (2)H/H of fat. The DFDM (2)H/H, and (18)O/(16)O values, excluding an outlier, are significantly correlated with the corresponding values in meteoric waters, thus allowing us to trace the variability of geoclimatic factors. (15)N/(14)N is influenced by pedoclimatic conditions, whereas (34)S/(32)S is influenced by the sea spray effect and the surface geology of the provenance area. By applying stepwise linear discriminant analysis only the (2)H/H of fat was found not to be significant and 97.7% of the samples were correctly assigned to the lamb type and more than 90% cross-validated. With the feeding regime, 97.7% of the samples were both correctly assigned and cross-validated using a predictive model including (13)C/(12)C, (15)N/(14)N, (18)O/(16)O, (34)S/(32)S of DFDM and (18)O/(16)O of fat.

H, C, N and S stable isotopes and mineral profiles to objectively guarantee the authenticity of grated hard cheeses.

In compliance with the European law (EC No. 510/2006), geographical indications and designations of origin for agricultural products and foodstuffs must be protected against mislabelling. This is particularly important for PDO hard cheeses, as Parmigiano Reggiano, that can cost up to the double of the no-PDO competitors. This paper presents two statistical models, based on isotopic and elemental composition, able to trace the origin of cheese also in grated and shredded forms, for which it is not possible to check the logo fire-marked on the rind. One model is able to predict the origin of seven types of European hard cheeses (in a validation step, 236 samples out of 240 are correctly recognised) and the other specifically to discriminate the PDO Parmigiano Reggiano cheese from 9 European and 2 extra-European imitators (260 out of 264 correct classifications). Both models are based on Random Forests. The most significant variables for cheese traceability common in both models are δ(13)C, δ(2)H, δ(15)N, δ(34)S and Sr, Cu, Mo, Re, Na, U, Bi, Ni, Fe, Mn, Ga, Se, and Li. These variables are linked not only to geography, but also to cow diet and cheese making processes.

Traceability along the production chain of Italian tomato products on the basis of stable isotopes and mineral composition.

The paper shows the variability of stable isotope ratios and mineral composition in tomato and derivatives along the production chain (juice, passata and paste) in order to evaluate the possibility of tracing their geographical origin. The ratios (13)C/(12)C, (15)N/(14)N, (18)O/(16)O, D/H, (34)S/(32)S and the content of Li, Be, B, Na, Mg, Al, P, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Y, Mo, Ag, Cd, Sn, Sb, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Dy, Ho, Tm, Yb, Ir, Tl, Pb, U and of nitrates, chlorides, sulphates and phosphates were measured by Isotope Ratio Mass Spectrometry, Inductively Coupled Plasma Mass Spectrometry and Ion Chromatography, respectively. The tomato products were from three Italian regions - Piedmont, Emilia Romagna, and Apulia. By applying linear discriminant analysis on 17 of these parameters (Gd, La, Tl, Eu, Cs, Ni, Cr, Co, δ(34)S, δ(15)N, Cd, K, Mg, δ(13)C, Mo, Rb and U) excellent discrimination among products from the three regions was achieved. Irrespective of the processing technology, over 95% of the samples were correctly reclassified in cross-validation into the production site. The use of these parameters will allow the development of analytical control procedures that can be used to check the geographical provenance of Italian tomatoes and products derived from them.

Stable isotope ratios of carbon and hydrogen to distinguish olive oil from shark squalene-squalane

Squalene and its hydrogenated derivate squalane are widely used in the pharmaceutical and cosmetic fields. The two compounds are mainly produced from the liver oil of deep sea sharks and from olive oil distillates. Squalene and squalane from shark cost less than the same compounds derived from olive oil, and the use of these shark-derived compounds is unethical in cosmetic formulations. In this work we investigate whether (13)C/(12)C and (2)H/(1)H ratios can distinguish olive oil from shark squalene/squalane and can detect the presence of shark derivates in olive oil based products. The (13)C/(12)C ratios (expressed as delta(13)C values) of bulk samples and of pure compounds measured using isotope ratio mass spectrometry (IRMS) were significantly lower in authentic olive oil squalene/squalane (N: 13; -28.4 +/- 0.5 per thousand; -28.3 +/- 0.8 per thousand) than in shark squalene/squalane samples (N: 15; -20.5 +/- 0.7 per thousand; -20.4 +/- 0.6 per thousand). By defining delta(13)C threshold values of -27.4 per thousand and -26.6 per thousand for olive oil bulk and pure squalene/squalane, respectively, illegal addition of shark products can be identified starting from a minimum of 10%. (2)H/(1)H analysis is not useful for distinguishing the two different origins. Delta(13)C analysis is proposed as a suitable tool for detecting the authenticity of commercial olive oil squalene and squalane samples, using IRMS interfaced to an elemental analyser if the purity is higher than 80% and IRMS interfaced to a gas chromatography/combustion system for samples with lower purity, including solutions of squalane extracted from cosmetic products.

Stable Isotope Ratio Analysis for Assessing the Authenticity of Food of Animal Origin

The main elemental constituents (H, C, N, O, and S) of bio-organic material have different stable isotopes (2 H, 1 H; 13 C,12 C; 15 N,14 N; 18 O,17 O,16 O; 36 S, 34 S, 33 S, and 32 S). Isotopic ratios can be measured precisely and accurately using dedicated analytical techniques such as isotope ratio mass spectrometry (IRMS). Analysis of these ratios shows potential for assessing the authenticity of food of animal origin. In this review, IRMS analysis of food of animal origin and variability factors related to stable isotope ratios in animals are described. The study also lists examples of application of stable isotope ratio analysis to meat, dairy products, fish, and shellfish and emphasizes the strengths and weaknesses of the technique. Geographical, climatic, pedological, geological, botanical, and agricultural factors affect the stable isotope ratios (SIR) of bio-elements, and SIR variations are ultimately incorporated into animal tissue through eating, drinking, breathing, and exchange with the environment, being recorded in the resulting foods. SIR analysis was capable of determining geographical origin, animal diet, and the production system (such as organic/conventional or wild/farmed) for pork, beef, lamb, poultry, milk, butter, cheese, fish, and shellfish. In the case of the hard PDO (protected designations of origin) cheeses Grana Padano and Parmigiano Reggiano it is also used in real-life situations to assess the authenticity of grated and shredded cheese on the market.

Validation of methods for H, C, N and S stable isotopes and elemental analysis of cheese: results of an international collaborative study.

RATIONALE PDO cheeses, such as Parmigiano Reggiano and Grana Padano, which cost more than double generic similar cheeses, must be protected against mislabelling. The aim of this study was to validate the methods for the isotopic and elemental analysis of cheese, in order to support official recognition of their use in authenticity assessment. METHODS An international collaborative study based on blind duplicates of seven hard cheeses was performed according to the IUPAC protocol and ISO Standards 5725/2004 and 13528/2005. The H, C, N and S stable isotope ratios of defatted cheese determined using Isotope Ratio Mass Spectrometry (IRMS) and the content of Li, Na, Mn, Fe, Cu, Se, Rb, Sr, Mo, Ba, Re, Bi, U in cheese after acid microwave digestion using Inductively Coupled Plasma Mass Spectrometry or Optical Emission Spectrometry (ICP-MS or -OES) were measured in 13 different laboratories. RESULTS The average standard deviations of repeatability (sr) and reproducibility (sR) were 0.1 and 0.2 ‰ for δ(13)C values, 0.1 and 0.3 ‰ for δ(15)N values, 2 and 3 ‰ for δ(2)H values, and 0.4 and 0.6 ‰ for δ(34)S values, thus comparable with results of official methods and the literature for other food matrices. For elemental data, the average RSDr and RSDR values ranged between 2 and 11% and between 9 and 28%, respectively, consistent with methods reported by the FDA and in the literature for cheese. CONCLUSIONS The validation data obtained here can be submitted to the standardisation agencies to obtain official recognition for the methods, which is fundamental when they are used in commercial disputes and legal debates.

The use of IRMS, (1)H NMR and chemical analysis to characterise Italian and imported Tunisian olive oils.

Isotope Ratio Mass Spectrometry (IRMS), (1)H Nuclear Magnetic Resonance ((1)H NMR), conventional chemical analysis and chemometric elaboration were used to assess quality and to define and confirm the geographical origin of 177 Italian PDO (Protected Denomination of Origin) olive oils and 86 samples imported from Tunisia. Italian olive oils were richer in squalene and unsaturated fatty acids, whereas Tunisian olive oils showed higher δ(18)O, δ(2)H, linoleic acid, saturated fatty acids β-sitosterol, sn-1 and 3 diglyceride values. Furthermore, all the Tunisian samples imported were of poor quality, with a K232 and/or acidity values above the limits established for extra virgin olive oils. By combining isotopic composition with (1)H NMR data using a multivariate statistical approach, a statistical model able to discriminate olive oil from Italy and those imported from Tunisia was obtained, with an optimal differentiation ability arriving at around 98%.

Hydrogen and oxygen stable isotope fractionation in body fluid compartments of dairy cattle according to season, farm, breed, and reproductive stage.

Environmental temperature affects water turnover and isotope fractionation by causing water evaporation from the body in mammals. This may lead to rearrangement of the water stable isotope equilibrium in body fluids. We propose an approach to detect possible variations in the isotope ratio in different body fluids on the basis of different homoeothermic adaptations in varying reproductive stages. Three different reproductive stages (pregnant heifer, primiparous lactating cow, and pluriparous lactating cow) of two dairy cattle breeds (Italian Friesian and Modenese) were studied in winter and summer. Blood plasma, urine, faecal water, and milk were sampled and the isotope ratios of H (2H/1H) and O (18O/16O) were determined. Deuterium excess and isotope-fractionation factors were calculated for each passage from plasma to faeces, urine and milk. The effects of the season, reproductive stages and breed on δ 2H and δ 18O were significant in all the fluids, with few exceptions. Deuterium excess was affected by season in all the analysed fluids. The correlations between water isotope measurements in bovine body fluids ranged between 0.6936 (urine-milk) and 0.7848 (urine-plasma) for δ 2H, and between 0.8705 (urine-milk) and 0.9602 (plasma-milk) for δ 18O. The increase in both isotopic δ values in all body fluids during summer is representative of a condition in which fractionation took place as a consequence of a different ratio between ingested and excreted water, which leads to an increased presence of the heavy isotopes. The different body water turnover between adult lactating cattle and non-lactating heifers was confirmed by the higher isotopic δ for the latter, with a shift in the isotopic equilibrium towards values more distant from those of drinking water.