How to guarantee authenticity and traceability of agri-food and supplements products thanks to the application of isotopic analysis of bioelements

Stable isotope ratio analysis of bio-elements (hydrogen, carbon, nitrogen, oxygen and sulphur) has been used since the 1990s to check food authenticity and traceability of a wide variety of food commodities (Rossmann, 2001). In the last few years, examples of applications also in the pharmaceutical and cosmetic field have been reported (Pellati et al., 2013; Perini et al., 2017, 2021; Perini, Paolini, et al., 2019; Perini, Pianezze, et al., 2019). The use of stable isotope analysis for products authentication purposes is possible thanks to isotopic fractionation occurring in several processes and reactions (biological, biochemical, physical, chemical etc.) which generates unique isotopic signatures. For this reason, the application of this technique on the bulk samples as well as on specific components (e.g. aroma compounds) can be used to detect the origin of an ingredient (synthetic or natural), the substitution of one ingredient for another, as well as the geographical and/or botanical origin of the products. The widespread and well-known technique based on the coupling between elemental analyzer and mass spectrometer (EA-IRMS) is now flanked by liquid chromatography (LC-IRMS) and gas chromatography (GC-IRMS). Today it is therefore possible to analyze not only the bulk of the matrices but also their individual components. The δ13C and δ2H values of vanillin can determine whether the product is natural (deriving from the expensive CAM plant Vanilla), biotechnologically derived or synthetic (Perini, Pianezze, et al., 2019). Moreover, the δ13C values of specific components of Rosa damascene mill., one of the most expensive essential oils in the market world, can indicate the fraudulent addition of cheaper oil from a C4 plant (e.g. Cymbopogon martinii, palmarosa) (Pellati et al., 2013). In pharmaceutical and cosmetic formulations, δ13C analysis is a suitable tool to discriminate between squalene and squalane from shark (illegal) and from olive oil (expensive) (Camin et al., 2010) as well as between monacolin K (contained in the fermented dietary supplement red yeast rice) and the commercially marketed statin, lovastatin (Perini et al., 2017).The L-theanine extracted from Camellia Sinensis is easily distinguishable from that obtained biosynthetically (Perini et al., 2021). It is possible to combine different isotopic signatures to guarantee the natural origin of curcumin, caffeine (Ding et al., 2019), tartaric acid and its derivatives. These examples demonstrate that the isotopic fingerprint represent an effective tool for the authenticity assessment of economically important pharmaceutical, cosmetic and supplement products

How to guarantee the natural origin of nutraceutical and pharmaceutical products? The potential of the stable isotope ratios analysis

The analysis of the ratio between the heavy and light isotopes of the elements carbon (13C/12C), nitrogen (15N/14N), sulfur (34S/32S), oxygen (18O/16O) and hydrogen (2H/1H) is well known for its power to discriminate the geographical origin and guarantee the authenticity of many agri-food products [1]. In recent years, the field of application of this technique has expanded to include nutraceuticals and pharmaceuticals, in particular in order to guarantee their natural origin. Chemically identical molecules are significantly different from an isotopic point of view due to the isotopic fractionation that occurs in different processes and reactions (biological, biochemical, physical, chemical, etc.) which generates unique isotopic signatures in the product synthesized by plants compared to that produced in the laboratory usually starting from fossil sources. Thanks to the coupling of isotopic mass spectrometry to liquid chromatography (LC-IRMS) and gas chromatography (GC-IRMS) it is now possible to discriminate between natural and/or synthetic origin not only of the bulk product but also of its specific components. The "Compound specific analysis" makes it possible to identify much more sophisticated frauds than in the past such as, for example, the addition of a single synthetic component to a natural substrate in order to artificially increase its strength. In this context, the δ13C analysis is a suitable tool to discriminate between Monacolin K (contained in red yeast rice-based dietary supplements) and the marketed statin [2] and between natural L-theanine (extracted from Camellia Sinensis) and the biosynthetically produced one [3]. The isotope ratios of hydrogen and, in some cases, carbon exhibit significantly different ranges of variability between natural extracts (such as curcuminoids [4] and cannabidiols [5]) and their synthetic adulterant, allowing for the identification of not only the two origins, but also the fraudulent additions of synthetic products to the natural complex (spiked samples). The combination of GC-MS/MS and GC-IRMS is potentially useful for botanical classification between lavender (Lavandula angostifolia) and lavandin (Lavandula hybrida) essential oils thus representing an additional powerful tool for assessing the authenticity of commercial essential oil

High density balsamic vinegar: application of stable isotope ratio analysis to determine watering down

Aceto balsamico di Modena IGP (ABM) is an Italian worldwide appreciated PGI (Protected Geographical Indication) vinegar, obtained from cooked and/or concentrated grape must (at least 20% of the volume), with the addition of at least 10% of wine vinegar and a maximum 2% of caramel for color stability (EU Reg. 583/ 2009). The geographical origin of ABM ingredients is never specified. Since 2013, the European Committee for Standardization (CEN) has issued a method for determining the water fraudulently added to the vinegar and the balsamic vinegar product (EN16466-3 18O-IRMS). The method is based on the stable isotope ratios analysis of the bulk AMB sample (expressed as δ18O in ‰ with respect to the international standard V-SMOW2). Balsamic vinegars with very high density (higher than 1.37 g / mL of sugar) are available on the market. They are obtained by adding a high amount of concentrated must or by a long aging of the product in the barrel, which leads to an intense evaporation and concentration. Products with such high density cannot be analyzed by using the official method as reported in the EN16466-3 18O-IRMS. Indeed, in this conditions, the equilibration between CO2 and the water in the sample, being the base principle of the process, does not occur. In this work, the official method has been modified and validated, calculating repeatability (r) and reproducibility (R), by proceeding with a prior dilution of the sample and by applying a correction to the data in order to eliminate the diluent isotopic contribution. Considering the limit value of δ18O for a non-watered product reported in the literature for vinegar and for rectified concentrated must [1-2], the threshold limit of δ18O below which the ABM product can be considered as adulterated was identified. References [1] J. Agric. Food Chem. 2014, 62, 32, 8197–8203 [2] Food Control 2013, 29(1), 107–11

Authentication and geographical characterisation of Italian grape musts through glucose and fructose carbon isotopic ratios determined by LC IRM

The authenticity of grape musts is normally checked through the stable isotopic analysis of carbon (δ13C) after fermentation and distillation by following the official OIV MA AS-312-06 method. Unfortunately, it presents some issues that are difficult to over come. Grape must samples can only be analysed after they have been fermented to obtain ethanol. The process must be carried out under careful control of the fermentation to avoid the presence of unwanted by-products arising from a premature fermentation interruption. Moreover, if the musts have been preserved by the addition of sulphur dioxide (SO2 ), they must undergo an additional step to eliminate the SO2 , which would affect the fermentation. Once the product has been fermented, the ethanol must be separated using specific distillation columns (such as the Cadiot ones) making it possible to obtain ethanol free of isotopic fractionation with a minimum alcohol degree of 95% vol. In this study, the alternative use of a technique based on δ13C isotopic analysis of the major sugars of the grape must by liquid chromatography coupled with isotope ratio mass spectrometry (LC-IRMS) is provided. In LC–IRMS, analytes are separated on an LC system and consecutively oxidized in an online reactor to CO2 , which is required for the determination of compound-specific carbon isotopic ratios. This technique has been already used in the study of matrices such as wine [1], ethanol [1,2], glycerol [2], and honey [3] to detect fraudulent alterations of their natural composition such as the addition of exogenous sugars to the products. The LC-IRMS allows a single separation of the individual components of a sample and makes it possible to determine their δ13C values online, avoiding both the disadvantages of off-line methods and the disadvantages of methods requiring a derivatization step (such as GC-C-IRMS), causing the addition of extra carbons. In order to discriminate between musts from different areas of Italy, a preliminary dataset was considered; the δ13C isotopic ratios of glucose and fructose of around 100 authentic Italian must samples from 16 different sampling regions were analysed. In addition, the δ13C variability in authentic and fake must (added with increasing percentages of exogenous sugars) has been explored and tested to verify their validity as fraud detectors. The two analysed parameters, ranging from −29.8‰ to −21.9‰, are well correlated (R2 = 0.7802) and the northern Italian regions showed significantly more negative δ13C values for both sugars than the rest of the dataset (Figure 1). By using the LC-IRMS technique, the addition of exogenous sugars, such as fructose and glucose from C4 photosynthetic cycle plants, is easily detectable as it modifies the δ13C of the individual sugar

Fatty Acid and Multi-Isotopic Analysis (C, H, N, O) as a Tool to Differentiate and Valorise the Djebel Lamb from the Mountainous Region of Tunisia

The objective of this study was to distinguish between the Tunisian Djebel lamb meat and meat from typical Tunisian production systems (PSs) through the fatty acids (FAs) profile and the stable isotope ratio analysis (SIRA). Thirty-five lambs from three different regions and PSs (D = Djebel, B = Bou-Rebiaa, and O = Ouesslatia) were considered for this purpose. The results demonstrated that the PS and the geographic origin strongly influenced the FA profile of lamb meat. It was possible to discriminate between the Djebel lamb meat and the rest of the dataset thanks to the quantification of the conjugated linoleic acids (CLA) and the branched chain FAs. Moreover, statistically different concentrations of saturated, monounsaturated and polyunsaturated FAs and a different n-6/n-3 ratio were found for grazing (D and BR) and indoor (O) lambs, making it possible to discriminate between them. As for the stable isotope ratio analysis, all parameters made it possible to distinguish among the three groups, primarily on the basis of the dietary regimen (δ(13C) and δ(15N)) and breeding area (δ(18O) and δ(2H))

Chitosan in wine industry: identification of origin by a multidisciplinary approach and application in oenology

Aim: Chitosan is a promising antimicrobial agent, capable of providing control of a wide range of spoilage microorganisms. To guarantee the fugal origin of chitosan, the only authorized for oenological practices, a multidisciplinary approach based on the measurement of the stable isotope ratios, Fourier transform infrared spectrometry and thermogravimetric analysis was proposed. In addition, the activity of chitosan against food related microorganisms was evaluated by different experiments aimed to discriminate between chemical and physical action of chitosan vs. the microorganisms. Method: The measurement of the stable isotope ratios (SIR) of carbon δ13C, nitrogen δ15N, oxygen δ18O and hydrogen δ2H of 35 samples of chitosan and the data of maximum degradation temperatures (obtained by TGA) combined with those of the peak areas of amide I and NH2/Amide II (obtained by FTIR) were employed to discriminate chitosan different sources (fungal grown on different substrates vs crustacean). The antimicrobial activity was tested in static and stirred conditions, in a synthetic media, using type strains of most common technological or spoilage microorganism. Viability was evaluated by Petri plate counts. The activity of the soluble portion of chitosan was checked by inoculating microorganisms in the media after chitosan removal. Results: The Kruskal-Wallis test showed that δ13C and δ18O were the most significant parameters able to classified chitosan into three different groups (from fungus grown on C3 photosynthetic cycle plant substrate, from fungus on C4 substrate and from crustacean). HCA and PCA analysis based on TGA, FTIR and SIR data successfully distributed the tested samples into informative clusters. Tests of chitosan antimicrobial activity highlighted the different sensitivity of microorganisms to chitosans, allowing selective control of spoilage agents. However, yeast and bacteria involved in fermentation were damaged by chitosan, and the synthetic media treated with this molecule showed a less fermentative aptitude. Conclusion: A robust analytical strategy for the correct identification of chitosan samples from crustaceans or fungi was presented, based on the observation that diverse biosynthetic pathways during the formation of the chitin influenced the isotopic composition of chitosan. Results of toxicity tests suggest that chitosan is a promising tool in fermented beverage production, but an in-depth study of the biochemical interaction between chitosan and food microorganisms is necessary

Endophytes from African Rice (Oryza glaberrima L.) Efficiently Colonize Asian Rice (Oryza sativa L.) Stimulating the Activity of Its Antioxidant Enzymes and Increasing the Content of Nitrogen, Carbon, and Chlorophyll.

Bacterial endophytes support the adaptation of host plants to harsh environments. In this study, culturable bacterial endophytes were isolated from the African rice Oryza glaberrima L., which is well-adapted to grow with poor external inputs in the tropical region of Mali. Among these, six N-fixer strains were used to inoculate O. glaberrima RAM133 and the Asian rice O. sativa L. cv. Baldo, selected for growth in temperate climates. The colonization efficiency and the N-fixing activity were evaluated and compared for the two rice varieties. Oryza sativa-inoculated plants showed a fairly good colonization efficiency and nitrogenase activity. The inoculation of Oryza sativa with the strains Klebsiella pasteurii BDA134-6 and Phytobacter diazotrophicus BDA59-3 led to the highest nitrogenase activity. In addition, the inoculation of ‘Baldo’ plants with the strain P. diazotrophicus BDA59-3 led to a significant increase in nitrogen, carbon and chlorophyll content. Finally, ‘Baldo’ plants inoculated with Kl. pasteurii BDA134-6 showed the induction of antioxidant enzymes activity and the maintenance of nitrogen-fixation under salt stress as compared to the unstressed controls. As these endophytes efficiently colonize high-yielding crop varieties grown in cold temperate climates, they become good candidates to promote their growth under unfavorable conditions

Applicazione degli isotopi stabili di bioelementi a supporto dell'analisi forense

Nelle indagini forensi una delle domande centrali è: da dove proviene il campione esaminato? Un importante strumento analitico che può aiutarci nel rispondere al quesito è rappresentato dall’analisi dei rapporti di isotopi stabili di biolementi (IRMS). Esaminando le minuscole variazioni nell’abbondanza relativa degli isotopi stabili degli elementi carbonio ( 13C/12C), azoto ( 15N/14N), zolfo ( 34S/32S), idrogeno ( 2H/1H), e ossigeno ( 18O/16O), misurata come rapporto ed espressa in delta (ad esempio δ 13C) “per mil” (‰) o in milliurey (mUr), è possibile scoprire una firma unica e nascosta, rivelando informazioni sulla fonte e sulla storia di un campione. La composizione isotopica può essere misurata tramite spettrometro di massa isotopica (IRMS) su campioni talquali, dopo combustione con un analizzatore elementare o pirolisi tramite un pirolizzatore, o sui suoi singoli componenti volatili (es. vanillina) o resi volatili tramite derivatizzazione (es. amminoacidi) e separati tramite tecnica gascromatografica GC o separati tramite cromatografia liquida LC (es. zuccheri). Oggi questa analisi è uno strumento approvato nella scienza forense e viene utilizzata per un'ampia gamma di applicazioni. Essa può aiutare a determinare se campioni di sostanze chimicamente simili (farmaci, esplosivi, fibre, ecc.) condividono una fonte o una storia comune [1], viene utilizzata per distinguere i prodotti contraffatti (ad esempio prodotti farmaceutici, alimenti e aromi) da materiali autentici [2, 3], per determinare le fonti di tessuti animali come l'avorio, per indagare sui resti umani quando viene trovato un corpo non identificato [4] e per determinare se un atleta ha utilizzato farmaci per migliorare le sue prestazioni

Stable isotope ratio analysis to assess pharmaceuticals, cosmetics and dietary supplements authenticity

The stable isotope ratio analysis of the mayor bio-elements (hydrogen, carbon, nitrogen, oxygen and sulphur) makes it possible to authenticate pharmaceuticals, cosmetics and dietary supplements. This technique, applied to bulk samples and/or to specific compounds, can be used to detect the origin of an ingredient (synthetic or natural), the substitution of one ingredient with another, as well as the geographical and/or botanical origin of the products. The δ 13C and δ 2H values of vanillin can determine whether this product is natural (deriving from the expensive CAM plant Vanilla), biotechnologically produced or synthetic [1]. Moreover, the δ 13C values of specific components of Rosa damascene mill., one of the most expensive essential oils in the global market, can indicate the fraudulent addition of cheaper oil from C4 plants (e.g., Cymbopogon martinii, palmarosa) [2]. Finally, the δ 13C analysis is a suitable tool to discriminate between Monacolin K (contained in red yeast rice-based dietary supplements) and the marketed statin [3] and between natural L-theanine (extracted from Camellia Sinensis) and the biosynthetically produced one [4]. These examples show that the isotopic fingerprint represents an effective tool for the authenticity assessment of economically relevant pharmaceuticals, cosmetics and dietary supplement