Luca Ziller

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.

Compound-Specific δ15N and δ13C Analyses of Amino Acids for Potential Discrimination between Organically and Conventionally Grown Wheat

We present a study deploying compound-specific nitrogen and carbon isotope analysis of amino acids to discriminate between organically and conventionally grown plants. We focused on grain samples of common wheat and durum wheat grown using synthetic nitrogen fertilizers, animal manures, or green manures from nitrogen-fixing legumes. The measurement of amino acid δ(15)N and δ(13)C values, after protein hydrolysis and derivatization, was carried out using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Our results demonstrated that δ(13)C of glutamic acid and glutamine in particular, but also the combination of δ(15)N and δ(13)C of 10 amino acids, can improve the discrimination between conventional and organic wheat compared to stable isotope bulk tissue analysis. We concluded that compound-specific stable isotope analysis of amino acids represents a novel analytical tool with the potential to support and improve the certification and control procedures in the organic sector.

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.

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.

Intra-annual Pattern of Photosynthesis, Growth and Stable Isotope Partitioning in a Poplar Clone Subjected to Ozone and Water Stress

An experiment in open-top chambers was carried out in summer 2008 in Curno (northern Italy) in order to study the effects of ozone and drought stress on net photosynthesis, growth and stable isotope partitioning on cuttings of an ozone-sensitive poplar clone (Oxford). The biomass (as dry weight) of stems, leaves and roots was assessed five times during the growing season on a set of plants intended for destructive measurements (set 1). Another set of plants (set 2) was used for repeated measurements (net photosynthesis) and then destroyed at the end of the experiment. The dry weight of the stems in set 1 plants was calculated using allometric relations. The results showed that drought stress had a strong effect on all the parameters assessed. Ozone did not have any effect on biomass allocation in woody stems and stable isotope composition but reduced root/shoot ratios and caused loss of leaves during the growing season. The loss of leaves in the lower part of the crown was partly recovered with the emission of new young leaves in the upper part, thus restoring the overall photosynthetic apparatus. We conclude that the metabolic costs suffered to repair damage and support growth, and the reduction in starch reserves in the roots can compromise growth and the capacity to cope with stress factors in subsequent years.

Food web of a confined and anthropogenically affected coastal basin (the Mar Piccolo of Taranto) revealed by carbon and nitrogen stable isotopes analyses

Carbon and nitrogen stable isotope analysis was used to examine the food web of the Mar Piccolo of Taranto, a coastal basin experiencing several anthropogenic impacts. Main food sources (algal detritus, seaweeds, particulate organic matter (POM) and sediment organic matter (SOM)) and benthic and pelagic consumers were collected during two contrasting seasons (June and April), at four sites distributed over two inlets, and characterized by different level of confinements, anthropogenic inputs and the presence of mussels farming. δ13C values of organic sources revealed an important contribution of POM to both planktonic and benthic pathways, as well as the influence of terrigenous inputs within both inlets, probably due to high seasonal land runoff. Although δ13C of both sources and consumers varied little between sampling sites and dates, δ15N spatial variability was higher and clearly reflected the organic enrichment in the second inlet as well as the uptake of anthropogenically derived material by benthic consumers. On the other hand, within the first inlet, the isotopic composition of consumers did not change in response to chemical contamination. However, the impact of polluted sediments near the Navy Arsenal in the first inlet was detectable at the level of the macrobenthic trophic structure, showing high dominance of motile, upper level consumers capable to face transient conditions and the reduction of the more resident deposit feeders. We therefore underline the great potential of matching stable isotope analysis with quantitative studies of community structure to assess the effects of multiple anthropogenic stressors.

Studying trophic interactions between a plant pathogen and two different antagonistic microorganisms using a 13C‐labeled compound and isotope ratio mass spectrometry

RATIONALE The study of the interactions among microorganisms, especially between pathogens and other microorganisms, is a very useful way to identify possible biocontrol agents (BCAs). In this study we verified the capability of δ(13)C analysis using isotope ratio mass spectrometry (IRMS) to detect active parasitism or metabolic assimilation of (13)C-labeled Armillaria mellea (plant pathogen) by Trichoderma atroviride and Pseudomonas fluorescens (two BCAs). METHODS The three microorganisms were labeled in pure-culture using a specific medium to which D-glucose (13)C was added. The δ(13)C analysis of mycelia/cells and DNA was undertaken using IRMS at different times, to study the uptake kinetics of (13)C. The mechanisms of interaction were studied by implementing dual-culture tests and measuring the δ(13)C values of the two BCAs after 29  days of contact with the labeled pathogen. RESULTS A. mellea absorbed (13)C more slowly (plateau at 21 days) than T. atroviride and P. fluorescens (3 and 1 day, respectively) in pure-culture. The maximum δ(13)C values were higher in A. mellea and T. atroviride mycelia (8,019.9‰ and 10,383.7‰, respectively) than in P. fluorescens (953.4‰ in cells). In dual-culture the mycelia of T. atroviride which remained in direct contact with labeled A. mellea showed an increased δ(13)C value with respect to the unlabeled treatment (66.4‰ and -26.6‰, respectively), due to active interaction. Lower assimilation of (13)C was detected in P. fluorescens. CONCLUSIONS This work demonstrates that IRMS can be used for the in-depth study of direct parasitism and interaction process between biocontrol agents and labeled pathogens, allowing the screening of potential new BCAs.

Anthropogenic charcoal-rich soils of the XIX century reveal that biochar leads to enhanced fertility and fodder quality of alpine grasslands

Background and aimsSoil incorporation of charcoal (biochar) has been suggested as practice to sequester carbon, improve soil properties and crop yields but most studies have been done in the short term. Old anthropogenic charcoal-rich soils in the Alps enable to explore the long-term impact of charcoal addition to alpine grassland on seed germination, fertility and fodder nutritive value.MethodsA germination test and a growth experiment in pots with Festuca nigrescens Lam. and Trifolium pratense L. were performed using three different substrates: control soil (i.e. sandy-loam brown acid soils with some podsolization), charcoal hearth soil (i.e. charcoal-enriched anthropogenic soils derived from the carbonization of larch wood on flat terraces) and control soil mixed with a fraction of fresh larch wood charcoal to reach the soil-charcoal ratio of 0.6.ResultsBoth aged and fresh charcoal improved germination and markedly increased plant growth of the two plant species. The addition of fresh charcoal had an initial detrimental effect that disappeared in the second and third growth cycles. Plant Nitrogen:Phosphorus ratio revealed that growth was N-limited in the anthropogenic soils and P-limited in the control and freshly amended soils demonstrating that biochar aging is critical to obtain a significant growth stimulation. Plant nutrient contents revealed an improved fodder quality in both the charcoal amended soils.ConclusionsDespite the occurrence of limited toxic effects on seedlings, larch wood charcoal appears to have positive effects on fertility and fodder quality of alpine grasslands in the long term.

δ15N from soil to wine in bulk samples and proline

The feasibility of using δ(15) N as an additional isotopic marker able to link wine to its area of origin was investigated. The whole production chain (soil-leaves-grape-wine) was considered. Moreover, the research included evaluation of the effect of the fermentation process, the use of different types of yeast and white and red vinification, the addition of nitrogen adjuvants and ultrasound lysis simulating wine ageing. The δ(15) N of grapes and wine was measured in bulk samples and compounds, specifically in proline, for the first time. Despite isotopic fractionation from soil to wine, the δ(15) N values of leaves, grapes, wine and particularly must and wine proline conserved the variability of δ(15) N in the growing soil. Fermentation and ultrasound treatment did not affect the δ(15) N values of grape must, which was therefore conserved in wine. The addition of inorganic or organic adjuvants was able to influence the δ(15) N of bulk wine, depending on the amount and the difference between the δ(15) N of must and that of the adjuvant. The δ(15) N of wine proline was not influenced by adjuvant addition and is therefore the best marker for tracing the geographical origin of wine. Copyright