H OH metabolites OH Article Elemental and Isotopic Characterization of Tobacco from Umbria Luana Bontempo 1,*, Daniela Bertoldi 2, Pietro Franceschi 1, Fabio Rossi 3 and Roberto Larcher 2 1 Research and Innovation Centre, Fondazione Edmund Mach (FEM), Via E. Mach 1, 38098 San Michele all’Adige, Italy; [email protected] 2 Technology Transfer Centre, Experiment and Technological Services Department, Fondazione Edmund Mach (FEM), Via E. Mach 1, 38098 San Michele all’Adige, Italy; [email protected] (D.B.); [email protected] (R.L.) 3 Fattoria Autonoma Tabacchi Soc. Coop Agricola, Via G. Oberdan, 06012 Città di Castello, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-461-615-138 Abstract: Umbrian tobacco of the Virginia Bright variety is one of the most appreciated tobaccos in Europe, and one characterized by an excellent yield. In recent years, the Umbria region and local producers have invested in introducing novel practices (for production and processing) focused on environmental, social, and economic sustainability. Due to this, tobacco from Umbria is a leading commodity in the global tobacco industry, and it claims a high economic value. The aim of this study is then to assess if elemental and isotopic compositions can be used to protect the quality and geographical traceability of this particular tobacco. For the first time the characteristic value ranges of the stable isotope ratios of the bio-elements as a whole (δ2H, δ13C, δ15N, δ18O, and δ34S) and of the concentration of 56 macro- and micro-elements are now available, determined in Virginia Bright tobacco produced in two different areas of Italy (Umbria and Veneto), and from other worldwide Citation: Bontempo, L.; Bertoldi, D.; geographical regions. The ranges of variability of elements and stable isotope ratios had slightly Franceschi, P.; Rossi, F.; Larcher, R. different results, according to the three geographical origins considered. In particular, Umbria Elemental and Isotopic samples presented significantly lower content of metals potentially dangerous for human health. The Characterization of Tobacco from results of this first exploratory work highlight the possibility of characterizing tobacco from Umbria, Umbria. Metabolites 2021, 11, 186. and suggest widening the scope of the survey throughout Italy and foreign regions, in order to be https://doi.org/10.3390/ used to describe the geographical origin of tobacco in general and verify the origin of the products metabo11030186 on the market. Academic Editor: Peter Meikle Keywords: stable isotope ratios; elemental composition; tobacco; geographical origin Received: 11 February 2021 Accepted: 19 March 2021 Published: 22 March 2021 1. Introduction Nicotiana Publisher’s Note: MDPI stays neutral Tobacco is the common name of several plants of the genus (Solanaceae with regard to jurisdictional claims in family) and the general term for any product prepared from the cured leaves of the tobacco published maps and institutional affil- plant. Besides its hedonistic use, tobacco plays an important role in other sectors, and iations. has been increasingly investigated in the last few years [1]. Its alkaloid nicotine is widely used in agriculture as a contact insecticide [2], refined oil of tobacco seeds is used as edible oil [3,4] and has been proposed for the production of biodiesel [5], and tobacco seeds cake are used as feed for animals [6]. Last but not least, tobacco has been investigated for medical applications, and plays an important role in the advancement of plant biotechnology, as it Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. is an extremely versatile system for genetic manipulation and tissue culture research [6–11]. This article is an open access article Tobacco is currently grown in 12 European countries. The main producers are Italy, distributed under the terms and Spain, Poland, Greece, Croatia, France, Hungary, and Bulgaria, accounting for 99% of conditions of the Creative Commons the European Union’s (EU’s) tobacco production [12]. The European Union produces Attribution (CC BY) license (https:// around 200,000 tonnes of tobacco per year (data of 2019 from FAOSTAT—Statistic by creativecommons.org/licenses/by/ The Food and Agriculture Organization, accessed on 4 March 2021). The most popu- 4.0/). lar tobacco variety in the world is the Virginia Bright flue-cured tobacco, because of its Metabolites 2021, 11, 186. https://doi.org/10.3390/metabo11030186 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, 186 2 of 12 unique—and easily replicated—cultivation and curing methods, and it is a leading com- modity in the global tobacco industry. In particular, flue-cured Virginia varieties account for 71% of output, while light-air-cured Burley varieties are 16%, sun-cured or orien- tal 7%, and other varieties (dark-air-cured and fire-cured) 6% [12]. Italy is the leading producer of raw tobacco in the European Union, and among the 10 leading tobacco- producing countries worldwide (data from the Italian Ministry of Agriculture and the European Commission EC [12,13]). In Italy, 97% of tobacco is grown in only four regions: Campania, Umbria, Veneto, and Tuscany. The aromatic Virginia Bright, considered the best Italian and European flue-cured tobacco [14], is grown in the Veneto region, whereas the Virginia Filler variety, one of the most appreciated tobaccos in Europe and one char- acterized by an excellent yield, comes from the Umbria region. In the last few years, in general but in particular in Umbria, producers have tended to offer a high-quality product with a transparent and short supply chain, cultivated with the least possible impact on the surrounding environment, in compliance with the good agricultural practices proposed by CORESTA (Cooperation Centre for Scientific Research Relative to Tobacco) and with the agri-environmental measures defined at the local and national and international levels (social responsibility). The Umbria region and local producers, in particular, have worked side-by-side in order to reduce the impact of this cultivation on the environment and on the health of workers, as well as the health of the entire communities living in the area, and produce a product of high quality. In order to offer products of higher qual- ity, their chemical characterization is of paramount importance, in particular to assess and quantify undesirable and harmful byproducts (e.g., toxic elements) directly from the Nicotiana tabacum leaves. Even if many studies have been conducted to characterize Nicotiana tabacum from the genetic and molecular points of view (e.g., [15,16]), to the best of our knowledge few papers have focused on the elemental characterization of Nicotiana tabacum leaves and plants [17–20]. To date, the majority of the chemical investi- gations have been limited to the assessment of the presence of toxic elements that can be inhaled or absorbed through cigarette smoke [21–23]. In order to support the quality assessment, the chemical characterization has to be coupled with analytical techniques that enable the characterization and traceability of the provenance of tobacco, and that can objectively guarantee it. In this area, however, up to now, very few studies focused on the development of analytical methods able to trace the geographical origin of tobacco [24,25]. Stable isotope ratios of bio-elements, as well as elemental composition, have been successfully used in the last 30 years to trace and verify the origin of several foodstuffs [26,27]. Isotopic fractionation occurs during physical and chemical processes, as well as along metabolic pathways, and for this reason, the different isotopic ratios vary according to photosynthetic and nitrogen cycles, pedological characteristics of soils, agricultural practices, and geographical origin. The elemental composition is mainly affected by soil geology and pedological characteristics, but can be also a consequence of contamination during processing. The combination of stable isotope analysis and elemental composition is expected to be a powerful tool for the assessment of the geographical origin, as has been already widely demonstrated, for example, in food traceability [28,29]. In the particular case of tobacco, only a few papers have been published on the application of stable isotope ratio and trace element profiles to the tracing of its geographical origin. As far as stable isotope ratios are concerned, only two studies were published on the geographical traceability of tobacco, to the best of our knowledge. In 1997, Jamin and colleagues [25] published a paper on the multi-element and multi-site isotopic analysis of purified nicotine extracted from tobacco leaves, concluding that the carbon and nitrogen isotopic composition (13C/12C, 15N/14N) and the site-specific deuterium content of nicotine can provide useful information on the geographical origin of tobacco. Similarly, Binette and colleagues [30] found that the combination of 2H/1H and 15N/14N ratios can be useful for identifying foreign counterfeits of Canadian cigarettes. To date, the elemental composition of tobacco has been investigated mainly to assess the presence of toxic elements that can be inhaled or absorbed through cigarette smoke [21–23]. Only one Metabolites 2021, 11, 186 3 of 12 paper has focused on the use of elements to differentiate between tobaccos from different origins. Kazi and colleagues [21], indeed, have determined the content in Al, As, Cd, Ni, and Pb of filler tobacco of different local and imported branded cigarettes sold in Pakistan. Furthermore, Judd and Swami [31] found different values of the isotopic ratios of lead for cigarette tobaccos grown in different regions. To date, the stable isotope ratios of H, C, N, O, and S, and the elemental composition of Nicotiana tabacum have never been extensively explored for their characterization and the verification of their origin. Therefore, the aim of this study is to fill in this gap and start exploring and determining the characteristic value ranges for elements and the stable isotope ratios of the main cultivated tobacco variety (Virginia Bright).