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Determination of glycerol carbon stable isotope ratio for the characterization of Italian balsamic vinegars
Journal of Food Composition and Analysis 69 (2018) 33–38
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Original research article
Determination of glycerol carbon stable isotope ratio for the characterization of Italian balsamic vinegars
T
Simona Sighinolfia, Ilaria Baneschib, Simona Manzinia, Lorenzo Tassia, Luigi Dallaib, ⁎ Andrea Marchettia, a b
University of Modena and Reggio Emilia, Department of Chemical and Geochemical Sciences, via G. Campi 103, 41125, Modena, Italy CNR-Ist. Geoscienze e Georisorse, via Moruzzi 1, 56124, Pisa, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords: Food composition Food analysis Balsamic vinegars ABTM ABM Glycerol Carbon isotope ratio GC-C-IRMS
The gas chromatographic-combustion-isotopic ratio mass spectrometry (GC-C-IRMS) approach was applied to determine the compound-specific 13C/12C isotopic ratio of glycerol in balsamic vinegars of Modena (Italy). In particular, Italian Protected Designation of Origin and Protected Geographical Indication balsamic vinegars, namely the traditionally made Aceto Balsamico Tradizionale di Modena (ABTM) and the industrial Aceto Balsamico di Modena (ABM) products, were analyzed and a first attempt at classification was carried out. The carbon isotopic ratio of the glycerol polyalcohol varies on the basis of origin, varietal or provenance; therefore the discriminating potentiality of this species might be useful to elucidate the balsamic vinegar production process. To do this, a preliminary study was conducted and several marketable products, ABTM and ABM type, were subjected to measurements in addition to samples coming from three ABTM cask series (batteria). Experimental results highlighted the peculiarities of the two different production processes, suggesting the use of the carbon isotopic ratio of glycerol as an additional tool for balsamic vinegar authentication.
1. Introduction
purposes (Calderone et al., 2004; Jung et al., 2006; Cabanero et al., 2010). Glycerol is a polyalcohol naturally formed in grape-derived products as a consequence of sugar fermentation by yeast and also, in some cases, by the presence of molds (Calderone et al., 2004; Lorenzini et al., 2012). It represents the most abundant by-product obtained from the alcoholic fermentation of sugars to ethanol. Moreover, owing to its ability of changing the mouthfeel properties of wines and vinegars or increasing the sugar-free extract, glycerol is sometimes fraudulently added in order to mask poor quality food (Pretorius, 2000). Although the biochemical pathway for glycerol production is nowadays well known (Wang et al., 2001), the prediction of its amount in the final product is rather difficult. In fact, many variables may influence the biochemical mechanisms and the related yields, such as type of yeast and environmental conditions, i.e. pH, temperature and the presence of nutrients (Scanes et al., 1998). Concerning the 13C/12C ratio of glycerol, Weber et al. (1997) demonstrated that the isotope ratio is directly dependent on the composition of the glucose substrate and on the metabolic pathway that leads to its production. Therefore, all the variables that influence the 13C/12C ratio of sugars, namely the C3, C4 or CAM carbon fixation pathway (O’Leary, 1988), the yeast strain, the process temperature, etc., as well
Food characterization, in terms of determination of the chemical constituents, represents one of the main topics in current scientific research (Georgiou and Danezis, 2017). European Commission regulations promote food quality and food safety, with increased surveillance of products with geographical designations and/or indications such as the Protected Designation of Origin, PDO or the Protected Geographical Indication, PGI (Regulation (EC) No 510/2006; EUR-Lex document 52011DC0436, July 14th, 2011 –). To this end, a large variety of analytical methodologies have been developed in order to detect possible commercial frauds and to improve food authenticity (Danezis et al., 2016). Starting from the pioneering works of Bender (1971) and Bricout (1973), measuring the stable isotope ratios of 2H, 13C and 18O in biomolecules of food, one of the most promising techniques used for food authentication is the compoundspecific isotope ratio analysis, useful in distinguishing botanical and geographical origins of food (Van Leeuwen et al., 2014). For example, the stable isotopes ratio analysis of acetic acid has been reported as an assurance technique for the authenticity of vinegar (Perini et al., 2014; Dordevic et al., 2012) and measurement of 13C/12C ratio of glycerol has been widely applied in wine authentication and for classification
⁎
Corresponding author at: Department of Chemical and Geological Sciences Via Giuseppe Campi 103, 41125, Modena, Italy. E-mail address: [email protected] (A. Marchetti).
https://doi.org/10.1016/j.jfca.2018.02.002 Received 19 July 2017; Received in revised form 24 January 2018; Accepted 7 February 2018 Available online 10 February 2018 0889-1575/ © 2018 Elsevier Inc. All rights reserved.
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blend is then added with a certain amount of wine vinegar in order to achieve a total acidity in the final product of 6%, expressed as v/v of acetic acid, as stated by the PGI rule. The liquid must be then aged at least three months inside barrels before commercialization (Regulation (EC) No 583/2009). Unlike ATBM, the ABM production rule does not impose any geographical origin on its raw materials. On the basis of the production process, it is clear that ABTM is characterized by the presence of a greater amount of glycerol with respect to ABM, since the polyalcohol, produced during the alcoholic fermentation, owing to its low vapor pressure, can accumulate in the product during the long aging period. A lower amount of glycerol is expected in ABM since, generally, no fermentation of the musts occurs and the polyalcohol should mainly come from the added wine vinegar fraction. The present work represents a preliminary study to test the potentialities of the 13C/12C carbon isotope ratio of glycerol as a tool for food authentication. This includes the development of an analytical procedure to measure the glycerol 13C/12C in balsamic vinegars, using GC-C-IRMS, and the characterization of balsamic vinegars in terms of glycerol content and 13C/12C values. Several marketable ABTM, “extravecchio” type, in addition to samples coming from three different ABTM cask series, namely BatA, BatB and BatC, were characterized. Moreover, ABM samples from different production years, 2008 to 2011, representative of the different producers of the Modena province, were also analyzed using the same variables.
as the climate and/or geography of the production area, also influence the 13C/12C ratio of glycerol. Fronza et al. (1998) reported that climatic conditions, due to seasonal or geographical variation, influence the glycerol 13C/12C in wines. In particular, wet and cold years lead to a depletion of the 13C of glycerol while dry and hot years lead to an enrichment of the same isotope. Moreover, the same authors found a direct correlation between 13C/12C of glycerol and 13C/12C of ethanol, from the same wine, specifically a depletion of 13C/12C in glycerol by 2.5–3.50/00 compared to ethanol, while other authors report a depletion of 3.6–4.10/00 (Cabanero et al., 2010 and references herein reported). These findings allow the definition of a 13C/12C scale for glycerol to be used for grape-derived products starting from the measured 13 C/12C of ethanol and vice versa. In addition, since glycerol can be obtained also from animal fats, the 13 C/12C ratio will account for the diet of the animals too. As a consequence, the possibility to discriminate between natural or synthetic glycerol, obtained from the chemical hydrolysis of vegetal/animal fats, from industrial synthesis or from the fermentation of sugars of different plants, represents a powerful tool to determine food quality and authenticity (Fronza et al., 1998). At present and to our knowledge, measurements of carbon isotopes of glycerol in balsamic vinegars, of both traditional and industrial origin, are not reported in literature. Balsamic vinegars, such as Aceto Balsamico Tradizionale di Modena, ABTM and Aceto Balsamico di Modena, ABM, represent interesting food matrices to investigate owing to their peculiar food chain processes. In particular, the European Commission, in 2000, awarded ABTM with the PDO recognition and in 2009 registered the PGI protection for ABM (Regulation (EC) No 813/ 2000; Regulation (EC) No 583/2009). Several analytical methods have been employed for the chemical characterization of these products and in particular for ABTM (Consonni et al., 2008a). Furthermore, the type and amount of characteristic metabolic by-products of Acetobacter fermentation, present in vinegar, have been used as origin and authenticity proof for vinegars (Belitz and Grosch, 1992; Consonni et al., 2008b; Papotti et al., 2015) and as quality markers (Giudici et al., 2009). The ABTM production chain, as reported in the production rule (ABTM PDO production rule) starts from the alcoholic fermentation of condensed cooked musts, obtained from selected grapes, coming from the Modena province and successive acetic bio-oxidation of the produced ethanol. The procedure for making ABTM is briefly described to better understand the peculiarities of the product chain process. Cooked must is the raw starting material for making ABTM. It is obtained from a must of selected grapes that is condensed by simmering gently over an open fire in uncovered pans. The aging process is carried out in a set of barrels composed of a variable number of wooden casks, generally from 5 to 10, of different volumes and made of different woods. During the aging process the liquid in each cask is kept constant by transferring a certain amount of vinegar from one cask to another in a decreasing progression. This procedure is called “topping up”. The first operation consists of taking from the oldest cask an aliquot of aged balsamic vinegar, which is marketed as ABTM inside the typical 100 mL bottle designed by Giugiaro and authorized by the ABTM Consortium. From the next oldest cask, vinegar is added to the oldest one in order to replace the volume that is lost. This procedure goes on by topping up one cask from the neighboring one until the youngest cask is reached. This one is then fed with the new cooked must (Cocchi et al., 2002). The vinegar is aged inside the barrels until it reaches the organoleptic features of the PDO rule: “affinato”,12 years of ageing, and “extravecchio”, 25 years of ageing (Cocchi et al., 2007). For Aceto Balsamico di Modena, ABM, the starting raw material generally consists of condensed grape musts obtained by multiple effects concentrators operating under vacuum conditions and relatively low temperatures (40–60 °C). In some cases, producers can also use blends obtained by mixing condensed and cooked musts to obtain balsamic vinegars with a caramel-like taste. The condensed must or the
2. Materials and methods 2.1. Chemicals Two different brands of glycerol were supplied by Carlo Erba (Milan, Italy) and J.T.Baker-Avantor (Milan, Italy), respectively. KOH (38%), used for the basic hydrolysis of different types of fats, and 37% HCl, were purchased from Sigma-Aldrich (Milan, Italy). Ethanol (98%) was supplied by Fluka (Thermo Fisher Scientific, Milan, Italy). Solutions, sample dilution as well as HPLC mobile phase were always prepared by using high purity deionized water, type 1, obtained from a Milli Q 185Plus apparatus (Millipore, Bedford, MA). Physical and chemical parameters for Type 1 water comply with ASTM and ISO 3696 grade purity specification (ASTM D1193, 1999). 2.2. Samples To investigate the glycerol 13C/12C differences among the ABTM samples, aged at least 25 years (“extravecchio” type), and the ABM samples, the analytical procedure was performed on 20 samples of marketable ABTM and 92 samples of ABM of different production years: 2008 (15), 2009 (26), 2010 (25) and 2011 (26). Furthermore, to evaluate changes of the glycerol 13C/12C during the ABTM chain process, 25 samples coming from three different cask series, BatA(11), BatB (6), BatC(8), were also examined. Moreover, in order to evaluate possible fraudulent addition of the polyalcohol, glycerol samples of different origins were analyzed. In particular, as far as samples of natural origin are concerned, 5 samples of glycerol prepared by sugars fermentation of different cereals (malt barley, malt cider, 2 malt maize and malt rice), 2 samples obtained by hydrolysis of vegetal fats (olive oil and sunflower oil), and 2 samples coming from the hydrolysis of animal fats (butter and lard), were investigated. While, as glycerol of unknown origin, 2 samples of different commercial brands (A and B), were taken into consideration. Glycerol samples coming from fermentation processes were obtained from batch cultures of Saccharomyces cerevisiae growing on maize, barley and apple juice (cider) and of Saccharomyces bayanus growing on maize and rice. Glycerol samples coming from vegetal fats (olive oil and sunflower oil) and animal fats (butter and lard) were obtained by basic hydrolysis with concentrated KOH, successive filtration, neutralization with HCl 34
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and purification by HPLC technique.
R δ 13Csample ( 0/00 ) = ⎜⎛ s − 1⎟⎞ × 1000 R ⎝ st ⎠
2.3. Sample preparation
where Rs is the ratio of 13C/12C in the sample and Rst is the ratio of the international standard used. The result of this calculation is a relative δ (0/00) calibrated against the international standard. The certified IAEA-CH7 (polyethylene) and IAEA-CH6 (sucrose) with δ13CV-PDB of −32.15 and −10.45 respectively (0.05 and 0.040/00 standard deviation respectively, available from the International Atomic Energy Agency (IAEA)) were used to define a commercial glycerol as secondary working standard (δ13CV-PDB = −27.7 with a 0.10/00 standard deviation), to correct eventual drift. The δ13C value for the working reference was monitored, so that it did not differ by more than 0.50/00 from the admissible value. If it did, the spectrometry apparatus was checked and adjusted. Aliquots of 3 μL of sample solution, prepared as before mentioned, were injected with a split ratio of 1:20. A standard solution of glycerol was analyzed twice, each day, for δ13C glycerol, in order to check instrumental reproducibility. The precision of the measurement for glycerol was determined by repeating the analysis several times on the standard, on different days and under reproducibility conditions. The reproducibility was quite good, with a standard deviation associated to all data of 0.20/00 (n = 40). The accuracy of the method was tested by comparing the values obtained for the commercial glycerol via GC-C-IRMS method and EA-IRMS, in order to validate the methodology. Analysis shows very close agreement with a difference Δ δ (EA-GC) less than 0.10/00.
In order to quantify glycerol and to separate the corresponding fraction from the sample matrix for the subsequent GC-c-IRMS analysis, HPLC was used. Before HPLC analysis, all the samples were diluted with deionized water and filtered through a 0.2-μm disposable membrane. Different dilutions, prepared by weight, were made for each sample type as a function of the density of the investigated sample. ABM and ABTM samples were diluted 5 and 10 times, respectively. Glycerol fractions, from HPLC, were collected according to retention time (tR = 16 min) and successively dried under a nitrogen stream. Just before the GC injection, glycerol samples were re-dissolved with ethanol to obtain a final glycerol concentration of about 3 g L−1. The standard glycerol solution was prepared by diluting a commercial sample to the same final concentration. 2.4. Equipment and instrumental settings Samples were weighed using a Gibertini E42S analytical balance (Gibertini Elettronica, Milan, Italy) with a sensitivity of ± 0.1 mg. HPLC quantitation and separation of glycerol, on the ABTM and ABM samples, were performed with a Beckman System Gold apparatus (Beckman Coulter, Milan, Italy), equipped with a 116 model isocratic pump, a 156 RI detector, a 406 model analogue interface module and a Rheodyne injector with a 100 μL loop. An Aminex HPX-87C column (300 × 7.8 mm), (Bio-Rad Laboratories S.r.L., Milan, Italy) packed with a styrene divinylbenzene resin, 8% crosslinked, thermostated at 75 °C, was used. The chromatographic separation was carried out with water as mobile phase with a flow rate of 0.6 mL min−1. Reproducibility of the instrumental method was checked by injecting a standard solution of glycerol twice a day, the corresponding standard deviation was associated to all data, SD = 0.1 g kg−1 (n = 20). The determination of the carbon stable isotopes ratio, 13C/12C, of the glycerol was carried out using a Thermo Finnigan Trace GC Ultra (Bremen, Germany) equipped with an Alltech Heliflex AT-WAX column (30 m × 0.25 mm × 0.20 μm; Analytical Columns, Croydon, UK). Helium was used as carrier gas and separation was performed at 1.1 mL min−1 flow rate. Carbon isotope ratio measurement was performed on-line using a GC Combustion Interface III connected to a Thermo Finnigan Delta Plus XP IRMS (Bremen, Germany). The gas chromatographic conditions were set as follows: injection temperature 270 °C, oven temperature program: initial temperature 120 °C for 2 min, then the temperature was increased with a rate of 15 °C min−1 to a final value of 240 °C, final hold time 5 min. The combustion oven was kept at 940 °C, while the reduction oven temperature was maintained at 650 °C. The gas stream containing the CO2 molecules, obtained from the polyalcohol oxidation, enters the ion source of the mass spectrometer where the CO2 molecules are ionized, accelerated and separated in a magnetic sector analyzer. CO2 ions, coming from the polyalcohol oxidation, were measured by means of Faraday cups centered on the 44, 45 and 46 mass-to-charge (m/z) values, respectively.
2.6. Statistical analysis Data elaboration was carried out with PLS toolbox 8.2 (Eigenvector Research Inc., Wenatchee, WA) in Matlab 9.0 environment (MathWorks Inc.). An alpha level of confidence of 0.05 was used for all statistical tests. 3. Results and discussion 3.1. Glycerol characterization An investigation was carried out relative to some commercial glycerol samples (A and B) obtained from different suppliers, and on several glycerol samples of different natural origin. Table 1 reports the δ13C values measured by GC-C-IRMS that can be useful for a better understanding of the general trend of δ13C of glycerol in samples of different origin. Considering the reported data, as expected, the plant or animal origin of the sample influence the δ13C values. In particular, glycerol from C3 plants such as: sunflower, olive, barley, and rice, gives δ13C values ranging from −26 to −30‰, while for C4 plants, δ13C values are more positive and close to −18‰. Values of δ13C evaluated on glycerol samples of animal origin cover a wide range, probably Table 1 δ13C (0/00) data for glycerol samples of different varietal origin and brand, the corresponding standard deviation is 0.2 0/00. glycerol commercial A commercial B butter lard sunflower oil olive oil S. cerevisiae malt barley S. cerevisiae malt cider S. bayanus malt rice S. cerevisiae malt maize S. bayanus malt maize
2.5. Calibration and isotopic calculation At the beginning of each run, three pulses of CO2 reference gas were admitted into the inlet system for about 20 s. The constant flow rate during this period gives these peaks a flat-top appearance. A level of CO2 corresponding to 3–5 V at m/z 44 was used to calibrate the system. The 13C/12C abundance ratio was expressed as δ13C values calibrated against the international standard Vienna Pee Dee Belemnite (VPDB) (Fry, 2006). The delta notation is defined as: 35
carbon fixation pathway
δ13C
C3 C3 C3 C3 C3 C4 C4
−24.2 −28.6 −30.4 −17.8 −28.1 −30.4 −26.5 −28.9 −29.3 −17.6 −18.8
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distribution, with no significant differences among the years as confirmed by the ANOVA result (F(3,88) = 0.48, p-value = 0.70). However, different considerations can be emphasized by considering the δ13C data reported in Fig. 2 and ANOVA data analysis reported in Table 2a. In fact, based on the plot distribution of the experimental values, it is possible to highlight a decreasing trend of the ABM means data over the years. Moreover, the mean of the ABM2008 distribution, δ13C = −27.95‰, is significantly higher with respect to the values obtained for the ABM2009, δ13C = −29.32‰, ABM2010, δ13C = −29.91‰ and ABM2011, δ13C = −30.4‰, as confirmed by the ANOVA results. Considering that the glycerol in ABM mainly comes from the wine vinegar fraction added to the product and that no limitation on the geographical origin of this raw material is imposed, the observed variability of the data is probably in accordance with a nonspecific geographical origin of the wine vinegar, namely wine vinegars coming from different countries with different climatic conditions (inter-variability), in addition to climatic changes between years (intravariability) (Gaudillere et al., 2002; Martin et al., 1999). Therefore, to better characterize the ABM product a long-term investigation and the creation of a dedicated database are suggested.
depending on the diet of the animal and the fractionation mechanisms that take place in the animal’s metabolism. As an example, the δ13C of lard is close to that of maize malt. These experimental data agree well with previously reported results (Fronza et al., 1998). Moreover, glycerol from maize malt produced by S. cerevisiae shows a higher δ13C value than that produced by S. bayanus, supporting evidence that different yeast strains may influence carbon isotopic composition of glycerol, as found for wine glycerol by Calderone et al. (2004). As far as δ13C values determined on commercial samples of glycerol (unknown origin) are concerned, these lay inside the variability range of animal and plant values. From a first glimpse of the data of Table 1 it is evident that several factors affect the δ13C values and, as a consequence of these overlaps, the possibility to differentiate sample origin/varietal/exogenous addition or provenance is rather difficult only based on this variable alone. 3.2. ABM samples To better understand the discriminating potential of glycerol δ13C, several ABM vinegars, coming from different producers in the province of Modena (Italy) and sampled in different production years from 2008 to 2011, were investigated. The glycerol concentrations and δ13C results are reported, as box and whiskers plots, in Figs. 1 and 2, respectively. Owing to the peculiarities of the ABM production process, the concentration of glycerol in the commercial product depends on the wine vinegar added to the concentrated must. In fact, as expected, the average glycerol content in ABM varied from 1.5 to 8 g kg−1 and was lower than in ABTM where values ranged from 3.8 to 20 g kg−1 or more. This evidence is coherent with the assumption that the amount of glycerol in the concentrated must is almost negligible, since no fermentation processes occur during its production. However, the level of the polyalcohol may be higher in products obtained from juices coming from mould-infected grapes (Botrytis cinerea) where glycerol is produced in the grape berries during the ripening period (Lorenzini et al., 2012). Over the investigated years, the ABM samples show a constant trend of glycerol content both as concentration range and as interquartile
3.3. ABTM samples and cask series The analytical procedure was also applied to 20 ABTM samples and the experimental data are reported in graphical form in Figs. 1 and 2. Concerning the glycerol content, Fig. 1, the ABTM samples show a quite wide distribution that ranges from 8.7 to 22 g kg−1. Glycerol concentration ranges, typically, from 2 to 20 g kg−1 in wines and to 50 g kg−1 in aged traditional balsamic vinegars (Calderone et al., 2004; Turtura and Grasselli, 2003). The concentration values are in agreement with literature data and with the peculiarities of the ABTM chain process (alcoholic fermentation followed by acetic bio-oxidation and concentration effect during the long ageing period due to the topping up procedure and the loss of water). At the same time the glycerol content, evaluated along the investigated cask series, agrees well with the concentration range measured for the marketable ABTM. As a matter of fact, a direct comparison of the ABTM data with the BatA to BatC distributions is not possible since the casks contain an evolving
Fig. 1. box and whiskers plot of the glycerol concentration, g kg−1, measured for ABM, years 2008–2011, marketable ABTM and cask series BatA to BatC. The horizontal line inside the box represents the median value of the data; the dot represents the mean value. Circles represent the outliers.
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Fig. 2. box and whiskers plot of the δ13C of glycerol measured for ABM, years 2008–2011, marketable ABTM and cask series BatA to BatC. The horizontal line inside the box represents the median value of the data; the dot represents the mean value. Circles represent the outliers.
considered all of endogenous origin. The latter concerns BatC, where the average δ13C (−26.6 ± 1.4‰) is higher than in ABTM. The ANOVA (Table 2b) confirms that there is a meaningful difference between groups and, considering the plot of Fig. 2, it is clear to see that this is due to BatC samples distributions. Taking into account that the production rule for ABTM limits the use of raw materials, in terms of geographical origin, to grapes coming from the province of Modena and considering that seasonal variation is not so pronounced within the production area, it is quite surprising that BatC glycerol δ13C variability lay outside the variability range of the marketable ABTM. The most plausible reason for this behavior could be deviation from the traditional preparation of this cask series in terms of raw material, such as the use of mixtures of concentrated musts and wine vinegars of different geographical origins and/or varietals. This consideration is also supported by the evidence that the upper interquartile limit lies outside the above mentioned interval for vine plants, highlighting the hypothesis that some glycerol is derived from sugars of exogenous origin (Hattori et al., 2010). Moreover, considering the cut off value δ13Cethanol of −22‰ and the minimum δ13Cethanol of −30‰ observed for biotic C3 carbon fixation mechanism for vine plants (Christoph et al., 2015 and references herein reported; Smith and Epstein, 1971), and that glycerol is more depleted in δ13C as previously reported, the above interval for vine plants should be equivalent to a δ13C scale, measured for glycerol, that ranges from −25/–26 to −33/–34‰. As a consequence, ABTM data are coherent with grapes being used as the raw materials.
Table 2 One-way ANOVA to test for significant differences in ABM data (a) and ABTM data (b). a) source of variation
sum of squares
degree of freedom
F
F crit (α = 0.05)
between groups within groups
60.7126 65.9632
3 88
26.9984
2.70819
b) source of variation
sum of squares
degree of freedom
F
F crit (α = 0.05)
between groups within groups
56.09658 29.27586
3 41
26.18721
2.832747
product and, on the basis of the topping up procedure, only the glycerol content of the vinegar of the oldest barrels could be compared with the ABTM data (Cocchi et al., 2002). As regard the carbon isotope ratio, Fig. 2 reports the δ13C data evaluated on both the ABTM commercial products and the samples coming from the three cask series (BatA, BatB and BatC). Now, starting from the consideration that the δ13C value depends on the plant type and on the provenance of the grapes, it is possible to highlight that, for the ABTM commercial products, all the values are more or less distributed close to the average δ13C = −29.4 ± 0.5‰. Owing to the absence, to our knowledge, of other experimental data evaluated on the same matrix and considering the present numbers as face values, it is reasonably possible to assess that the glycerol, separated and measured in the ABTM, has the same natural origin. On the other hand, taking into account data obtained from the analysis of vinegars from three different cask series, two main conclusions can be drawn. The former is that BatA (δ13C = −29.3 ± 0.9‰) and BatB (δ13C = −29.9 ± 0.8‰) show average values coherent with the one determined on marketable ABTM. Hence there is no evidence of a particular trend inside the two series, confirming that the glycerol is formed during the alcoholic fermentation of grape sugars and it may be
4. Conclusion The use of the GC-C-IRMS approach, for the determination of δ13C of glycerol in balsamic vinegars of Modena, was a satisfactory technique for a deeper knowledge of this complex matrix. This preliminary study focuses on the evaluation of the potentialities of the isotopic methodology in discriminating glycerol of different sources and products with different chain processes, such as ABTM and ABM. In particular, glycerol concentration and δ13C results were coherent with grape-derived products for almost all of the investigated samples. Moreover, ABTM, due to its peculiar manufacturing process, shows a 37
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restricted δ13C variation for glycerol compared to the homologous industrial ABM. Nevertheless, taking into account the limited number of samples under investigation and despite the positive evidence obtained in the present study, the use of only one molecular marker was not sufficient to assess the authenticity of balsamic vinegars. In fact, in some cases the huge variability of the data may not allow discrimination of similar products or highlight fraudulent procedures. In conclusion, as seasonal and regional factors affect the level of isotope, data from other authentic balsamic vinegars have to be considered over a long-term investigation with the implementation of a dedicated data bank. Furthermore, monitoring more isotopic patterns in different molecules, e.g., acetic acid, in the case of vinegar or similar products, might enhance the diagnostic potential of the isotopic approach.
Fronza, G., Fuganti, C., Grasselli, P., Reniero, F., Guillou, C., Breas, O., Sada, E., Rossmann, A., Hermann, A., 1998. Determination of the 13C content of glycerol samples of different origin. Agric. Food Chem. 46, 477–480. Fry, B., 2006. Stable Isotope Ecology. Springer Verlag, New York, USA (ISBN: 0-38730513-0). Gaudillere, J.P., Leeuwen, C.V., Ollat, V., 2002. Carbon isotope composition of sugars in grapevine: on integrated indicator of vineyard water status. J. Exp. Bot. 53, 757–763. Georgiou, C.A., Danezis, G.P. (Eds.), 2017. Food Authentication, Management, Analysis and Regulation. John Wiley & Sons (ISBN 9781118810255). Giudici, P., Gullo, M., Solieri, L., Falcone, P.M., 2009. Technological and microbical aspects of Traditional Balsamic Vinegar and their influence on quality and sensorial properties. In: Taylor, Steve (Ed.), Advances in Food and Nutrition Research, V. 58. Academic Press (ISBN: 9780123744418). Hattori, R., Yamada, K., Shibata, H., Hirano, S., Tajima, O., Yoshida, N., 2010. Measurement of the isotope ratio of acetic acid in vinegar by HS-SPME-GC-TC/CIRMS. J. Agric. Food Chem. 58, 7115–7118. Jung, J., Jaufmann, T., Hener, U., Munch, A., Kreck, M., Dietrich, H., Mosandl, A., 2006. Progress in wine authentication: GC-C/P-IRMS measurements of glycerol and GC analysis of 2,3-butanediol stereoisomers. J. Agric. Food Chem. 223, 811–820. Lorenzini, M., Azzolini, M., Tosi, E., Zapparoli, G., 2012. Postharvest grape infection of botrytis cinerea and its interactions with other moulds under withering conditions to produce noble-rotten grapes. J. Appl. Microbiol. 114, 762–770. Martin, G.J., Mazure, M., Jouitteau, C., Martin, Y.L., Aguile, L., Allain, P., 1999. Characterization of the geographic origin of Bordeaux wines by a combined use of isotopic and trace element measurements. Am. J. Enol. Viticult. 50, 409–417. O’Leary, M.H., 1988. Carbon isotopes in photosynthesis. Bioscience 38 (5), 328–336. Papotti, G., Bertelli, D., Graziosi, R., Maietti, A., Tedeschi, P., Marchetti, A., Plessi, M., 2015. Traditional Balsamic vinegar and Balsamic vinegar of Modena analyzed by nuclear magnetic resonance spectroscopy coupled with multivariate data analysis. LWT- Food Sci. Technol. 60, 1017–1024. Perini, M., Paolini, M., Simoni, M., Bontempo, L., Vrhovsek, U., Sacco, M., Thomas, F., Jamin, E., Hermann, A., Camin, F., 2014. Stable isotope ratio analysis for verifying the authenticity of balsamic and wine vinegar. J. Agric. Food Chem. 62, 8197–8208. Pretorius, S.I., 2000. Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16, 675–729. Regulation (EC) No 510, 2006. On the protection of geographical indications and designations of origin for agricultural products and foodstuffs. Off. J. Eur. Union 93, 12–25 L93/12. (20 March 2006). Regulation (EC) No 583, 2009. Entering a name in the register of protected designations of origin and protected geographical indications [Aceto Balsamico di Modena (PGI)]. Off. J. Eur. Union 175, 7–11 L175/7. (3 July 2009). Regulation (EC) No 813, 2000. Supplementing the Annex to Commission Regulation (EC) No 1107/96 on the registration of geographical indications and designations of origin under the procedure laid down in Article 17 of Regulation (EEC) No 2081/92. Off. J. Eur. Union 100, 5–6 L100/43. (17 April 2000). Scanes, K.T., Hohrnann, S., Prior, B.A., 1998. Glycerol production by the yeast Saccharomyces cerevisiae and its relevance to wine: a review. S. Afr. J. Enol. Vitic. 19, 17–24. Smith, B.N., Epstein, S., 1971. Two categories of 13C/12C ratios for higher plants. Plant Physiol. 47, 380–384. Turtura, G.C., Grasselli, E.M., 2003. Caratteristiche microbiologiche e chimico-fisiche dell’Aceto Balsamico Tradizionale di Reggio Emilia. Industria delle bevande, XXXII Giugno. Van Leeuwen, K.A., Prenzler, P.D., Ryan, D., Camin, F., 2014. Gas chromatographycombustion-isotope ratio mass spectrometry for traceability and authenticity in foods and beverages. Compr. Rev. Food Sci. Food Saf. 13, 814–837. Wang, Z.-X., Zhuge, J., Fang, H., Prior, B.A., 2001. Glycerol production by microbial fermentation: a review. Biotechnol. Adv. 19, 201–223. Weber, D., Kexel, H., Schmidt, H.-L., 1997. 13C-pattern of natural glycerol: origin and practical importance. J. Agric. Food Chem. 45, 2042–2046.
References ABTM PDO production rule. http://ec.europa.eu/agriculture/quality/door/ documentDisplay.html?chkDocument=25_1_en. ASTM D1193, 1999. Standard Specification for Reagent Water, D19 Committee, Standard N0 7916. ASTM International, West Conshohocken, PA, USA. Belitz, H.-D., Grosch, W., 1992. Food Chemistry. Springer Ed, pp. 774–$9. Bender, M.M., 1971. Variations in the 13C/12C ratios of plants in relation to the pathway of photosynthetic carbon dioxide fixation. Phytochemistry 10 (6), 1239–1244. Bricout, J., 1973. Control of authenticity of fruit juices by isotopic analysis. J. AOAC Int. 56, 739–742. Cabanero, A.I., Recio, J.L., Rupérez, M., 2010. Simultaneous stable carbon isotopic analysis of wine glycerol and ethanol by liquid chromatography coupled to isotope ratio mass spectrometry. J. Agric. Food Chem. 58, 722–728. Calderone, G., Naulet, N., Guillou, C., Reniero, F., 2004. Characterization of european wine glycerol: stable carbon isotope approach. J. Agric. Food Chem. 52, 5902–5906. Christoph, N., Hermann, A., Wachter, H., 2015. 25 Years authentication of wine with stable isotope analysis in the European Union –review and outlook. Bio Web of Conferences 2, 02020. http://dx.doi.org/10.1051/bioconf/20150502020. Cocchi, M., Lambertini, P., Manzini, D., Marchetti, A., Ulrici, A., 2002. Determination of carboxylic acids in vinegars and in Aceto Balsamico Tradizionale di Modena by HPLC and GC Methods. J. Agric. Food Chem. 50, 5255–5261. Cocchi, M., Durante, C., Marchetti, A., Armanino, C., Casale, M., 2007. Characterization and discrimination of different aged ‘Aceto Balsamico Tradizionale di Modena’ products by head space mass spectrometry and chemometrics. Anal. Chim. Acta 589, 96–104. Consonni, R., Cagliani, L.R., Rinaldini, S., Incerti, A., 2008a. Analytical method for authentication of traditional Balsamic vinegar of Modena. Talanta 75, 765–769. Consonni, R., Cagliani, L.R., Benevelli, F., Spraul, M., Humpfer, E., Stocchero, M., 2008b. NMR and chemometric methods: a powerful combination for characterization of Balsamic and Traditional Balsamic Vinegar of Modena. Anal. Chim. Acta 611, 31–40. Danezis, G.P., Tsagkaris, A.S., Camin, F., Brusic, V., Georgiou, C.A., 2016. Food authentication: techniques, trends & emerging approaches. Trends Anal. Chem. 85, 123–132. Dordevic, N., Wehrens, R., Postma, G.J., Buydens, L.M.C., Camin, F., 2012. Statistical methods for improving verification of claims of origin for Italian wines based on stable isotope ratios. Anal. Chim. Acta 757, 19–25. Eur-lex document 52011DC0436 July 14th 2011. Green paper on promotion measures and information provision for agricultural products: a reinforced value-added European strategy for promoting the tastes of Europe.
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Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original research article
Determination of glycerol carbon stable isotope ratio for the characterization of Italian balsamic vinegars
T
Simona Sighinolfia, Ilaria Baneschib, Simona Manzinia, Lorenzo Tassia, Luigi Dallaib, ⁎ Andrea Marchettia, a b
University of Modena and Reggio Emilia, Department of Chemical and Geochemical Sciences, via G. Campi 103, 41125, Modena, Italy CNR-Ist. Geoscienze e Georisorse, via Moruzzi 1, 56124, Pisa, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords: Food composition Food analysis Balsamic vinegars ABTM ABM Glycerol Carbon isotope ratio GC-C-IRMS
The gas chromatographic-combustion-isotopic ratio mass spectrometry (GC-C-IRMS) approach was applied to determine the compound-specific 13C/12C isotopic ratio of glycerol in balsamic vinegars of Modena (Italy). In particular, Italian Protected Designation of Origin and Protected Geographical Indication balsamic vinegars, namely the traditionally made Aceto Balsamico Tradizionale di Modena (ABTM) and the industrial Aceto Balsamico di Modena (ABM) products, were analyzed and a first attempt at classification was carried out. The carbon isotopic ratio of the glycerol polyalcohol varies on the basis of origin, varietal or provenance; therefore the discriminating potentiality of this species might be useful to elucidate the balsamic vinegar production process. To do this, a preliminary study was conducted and several marketable products, ABTM and ABM type, were subjected to measurements in addition to samples coming from three ABTM cask series (batteria). Experimental results highlighted the peculiarities of the two different production processes, suggesting the use of the carbon isotopic ratio of glycerol as an additional tool for balsamic vinegar authentication.
1. Introduction
purposes (Calderone et al., 2004; Jung et al., 2006; Cabanero et al., 2010). Glycerol is a polyalcohol naturally formed in grape-derived products as a consequence of sugar fermentation by yeast and also, in some cases, by the presence of molds (Calderone et al., 2004; Lorenzini et al., 2012). It represents the most abundant by-product obtained from the alcoholic fermentation of sugars to ethanol. Moreover, owing to its ability of changing the mouthfeel properties of wines and vinegars or increasing the sugar-free extract, glycerol is sometimes fraudulently added in order to mask poor quality food (Pretorius, 2000). Although the biochemical pathway for glycerol production is nowadays well known (Wang et al., 2001), the prediction of its amount in the final product is rather difficult. In fact, many variables may influence the biochemical mechanisms and the related yields, such as type of yeast and environmental conditions, i.e. pH, temperature and the presence of nutrients (Scanes et al., 1998). Concerning the 13C/12C ratio of glycerol, Weber et al. (1997) demonstrated that the isotope ratio is directly dependent on the composition of the glucose substrate and on the metabolic pathway that leads to its production. Therefore, all the variables that influence the 13C/12C ratio of sugars, namely the C3, C4 or CAM carbon fixation pathway (O’Leary, 1988), the yeast strain, the process temperature, etc., as well
Food characterization, in terms of determination of the chemical constituents, represents one of the main topics in current scientific research (Georgiou and Danezis, 2017). European Commission regulations promote food quality and food safety, with increased surveillance of products with geographical designations and/or indications such as the Protected Designation of Origin, PDO or the Protected Geographical Indication, PGI (Regulation (EC) No 510/2006; EUR-Lex document 52011DC0436, July 14th, 2011 –). To this end, a large variety of analytical methodologies have been developed in order to detect possible commercial frauds and to improve food authenticity (Danezis et al., 2016). Starting from the pioneering works of Bender (1971) and Bricout (1973), measuring the stable isotope ratios of 2H, 13C and 18O in biomolecules of food, one of the most promising techniques used for food authentication is the compoundspecific isotope ratio analysis, useful in distinguishing botanical and geographical origins of food (Van Leeuwen et al., 2014). For example, the stable isotopes ratio analysis of acetic acid has been reported as an assurance technique for the authenticity of vinegar (Perini et al., 2014; Dordevic et al., 2012) and measurement of 13C/12C ratio of glycerol has been widely applied in wine authentication and for classification
⁎
Corresponding author at: Department of Chemical and Geological Sciences Via Giuseppe Campi 103, 41125, Modena, Italy. E-mail address: [email protected] (A. Marchetti).
https://doi.org/10.1016/j.jfca.2018.02.002 Received 19 July 2017; Received in revised form 24 January 2018; Accepted 7 February 2018 Available online 10 February 2018 0889-1575/ © 2018 Elsevier Inc. All rights reserved.
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blend is then added with a certain amount of wine vinegar in order to achieve a total acidity in the final product of 6%, expressed as v/v of acetic acid, as stated by the PGI rule. The liquid must be then aged at least three months inside barrels before commercialization (Regulation (EC) No 583/2009). Unlike ATBM, the ABM production rule does not impose any geographical origin on its raw materials. On the basis of the production process, it is clear that ABTM is characterized by the presence of a greater amount of glycerol with respect to ABM, since the polyalcohol, produced during the alcoholic fermentation, owing to its low vapor pressure, can accumulate in the product during the long aging period. A lower amount of glycerol is expected in ABM since, generally, no fermentation of the musts occurs and the polyalcohol should mainly come from the added wine vinegar fraction. The present work represents a preliminary study to test the potentialities of the 13C/12C carbon isotope ratio of glycerol as a tool for food authentication. This includes the development of an analytical procedure to measure the glycerol 13C/12C in balsamic vinegars, using GC-C-IRMS, and the characterization of balsamic vinegars in terms of glycerol content and 13C/12C values. Several marketable ABTM, “extravecchio” type, in addition to samples coming from three different ABTM cask series, namely BatA, BatB and BatC, were characterized. Moreover, ABM samples from different production years, 2008 to 2011, representative of the different producers of the Modena province, were also analyzed using the same variables.
as the climate and/or geography of the production area, also influence the 13C/12C ratio of glycerol. Fronza et al. (1998) reported that climatic conditions, due to seasonal or geographical variation, influence the glycerol 13C/12C in wines. In particular, wet and cold years lead to a depletion of the 13C of glycerol while dry and hot years lead to an enrichment of the same isotope. Moreover, the same authors found a direct correlation between 13C/12C of glycerol and 13C/12C of ethanol, from the same wine, specifically a depletion of 13C/12C in glycerol by 2.5–3.50/00 compared to ethanol, while other authors report a depletion of 3.6–4.10/00 (Cabanero et al., 2010 and references herein reported). These findings allow the definition of a 13C/12C scale for glycerol to be used for grape-derived products starting from the measured 13 C/12C of ethanol and vice versa. In addition, since glycerol can be obtained also from animal fats, the 13 C/12C ratio will account for the diet of the animals too. As a consequence, the possibility to discriminate between natural or synthetic glycerol, obtained from the chemical hydrolysis of vegetal/animal fats, from industrial synthesis or from the fermentation of sugars of different plants, represents a powerful tool to determine food quality and authenticity (Fronza et al., 1998). At present and to our knowledge, measurements of carbon isotopes of glycerol in balsamic vinegars, of both traditional and industrial origin, are not reported in literature. Balsamic vinegars, such as Aceto Balsamico Tradizionale di Modena, ABTM and Aceto Balsamico di Modena, ABM, represent interesting food matrices to investigate owing to their peculiar food chain processes. In particular, the European Commission, in 2000, awarded ABTM with the PDO recognition and in 2009 registered the PGI protection for ABM (Regulation (EC) No 813/ 2000; Regulation (EC) No 583/2009). Several analytical methods have been employed for the chemical characterization of these products and in particular for ABTM (Consonni et al., 2008a). Furthermore, the type and amount of characteristic metabolic by-products of Acetobacter fermentation, present in vinegar, have been used as origin and authenticity proof for vinegars (Belitz and Grosch, 1992; Consonni et al., 2008b; Papotti et al., 2015) and as quality markers (Giudici et al., 2009). The ABTM production chain, as reported in the production rule (ABTM PDO production rule) starts from the alcoholic fermentation of condensed cooked musts, obtained from selected grapes, coming from the Modena province and successive acetic bio-oxidation of the produced ethanol. The procedure for making ABTM is briefly described to better understand the peculiarities of the product chain process. Cooked must is the raw starting material for making ABTM. It is obtained from a must of selected grapes that is condensed by simmering gently over an open fire in uncovered pans. The aging process is carried out in a set of barrels composed of a variable number of wooden casks, generally from 5 to 10, of different volumes and made of different woods. During the aging process the liquid in each cask is kept constant by transferring a certain amount of vinegar from one cask to another in a decreasing progression. This procedure is called “topping up”. The first operation consists of taking from the oldest cask an aliquot of aged balsamic vinegar, which is marketed as ABTM inside the typical 100 mL bottle designed by Giugiaro and authorized by the ABTM Consortium. From the next oldest cask, vinegar is added to the oldest one in order to replace the volume that is lost. This procedure goes on by topping up one cask from the neighboring one until the youngest cask is reached. This one is then fed with the new cooked must (Cocchi et al., 2002). The vinegar is aged inside the barrels until it reaches the organoleptic features of the PDO rule: “affinato”,12 years of ageing, and “extravecchio”, 25 years of ageing (Cocchi et al., 2007). For Aceto Balsamico di Modena, ABM, the starting raw material generally consists of condensed grape musts obtained by multiple effects concentrators operating under vacuum conditions and relatively low temperatures (40–60 °C). In some cases, producers can also use blends obtained by mixing condensed and cooked musts to obtain balsamic vinegars with a caramel-like taste. The condensed must or the
2. Materials and methods 2.1. Chemicals Two different brands of glycerol were supplied by Carlo Erba (Milan, Italy) and J.T.Baker-Avantor (Milan, Italy), respectively. KOH (38%), used for the basic hydrolysis of different types of fats, and 37% HCl, were purchased from Sigma-Aldrich (Milan, Italy). Ethanol (98%) was supplied by Fluka (Thermo Fisher Scientific, Milan, Italy). Solutions, sample dilution as well as HPLC mobile phase were always prepared by using high purity deionized water, type 1, obtained from a Milli Q 185Plus apparatus (Millipore, Bedford, MA). Physical and chemical parameters for Type 1 water comply with ASTM and ISO 3696 grade purity specification (ASTM D1193, 1999). 2.2. Samples To investigate the glycerol 13C/12C differences among the ABTM samples, aged at least 25 years (“extravecchio” type), and the ABM samples, the analytical procedure was performed on 20 samples of marketable ABTM and 92 samples of ABM of different production years: 2008 (15), 2009 (26), 2010 (25) and 2011 (26). Furthermore, to evaluate changes of the glycerol 13C/12C during the ABTM chain process, 25 samples coming from three different cask series, BatA(11), BatB (6), BatC(8), were also examined. Moreover, in order to evaluate possible fraudulent addition of the polyalcohol, glycerol samples of different origins were analyzed. In particular, as far as samples of natural origin are concerned, 5 samples of glycerol prepared by sugars fermentation of different cereals (malt barley, malt cider, 2 malt maize and malt rice), 2 samples obtained by hydrolysis of vegetal fats (olive oil and sunflower oil), and 2 samples coming from the hydrolysis of animal fats (butter and lard), were investigated. While, as glycerol of unknown origin, 2 samples of different commercial brands (A and B), were taken into consideration. Glycerol samples coming from fermentation processes were obtained from batch cultures of Saccharomyces cerevisiae growing on maize, barley and apple juice (cider) and of Saccharomyces bayanus growing on maize and rice. Glycerol samples coming from vegetal fats (olive oil and sunflower oil) and animal fats (butter and lard) were obtained by basic hydrolysis with concentrated KOH, successive filtration, neutralization with HCl 34
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and purification by HPLC technique.
R δ 13Csample ( 0/00 ) = ⎜⎛ s − 1⎟⎞ × 1000 R ⎝ st ⎠
2.3. Sample preparation
where Rs is the ratio of 13C/12C in the sample and Rst is the ratio of the international standard used. The result of this calculation is a relative δ (0/00) calibrated against the international standard. The certified IAEA-CH7 (polyethylene) and IAEA-CH6 (sucrose) with δ13CV-PDB of −32.15 and −10.45 respectively (0.05 and 0.040/00 standard deviation respectively, available from the International Atomic Energy Agency (IAEA)) were used to define a commercial glycerol as secondary working standard (δ13CV-PDB = −27.7 with a 0.10/00 standard deviation), to correct eventual drift. The δ13C value for the working reference was monitored, so that it did not differ by more than 0.50/00 from the admissible value. If it did, the spectrometry apparatus was checked and adjusted. Aliquots of 3 μL of sample solution, prepared as before mentioned, were injected with a split ratio of 1:20. A standard solution of glycerol was analyzed twice, each day, for δ13C glycerol, in order to check instrumental reproducibility. The precision of the measurement for glycerol was determined by repeating the analysis several times on the standard, on different days and under reproducibility conditions. The reproducibility was quite good, with a standard deviation associated to all data of 0.20/00 (n = 40). The accuracy of the method was tested by comparing the values obtained for the commercial glycerol via GC-C-IRMS method and EA-IRMS, in order to validate the methodology. Analysis shows very close agreement with a difference Δ δ (EA-GC) less than 0.10/00.
In order to quantify glycerol and to separate the corresponding fraction from the sample matrix for the subsequent GC-c-IRMS analysis, HPLC was used. Before HPLC analysis, all the samples were diluted with deionized water and filtered through a 0.2-μm disposable membrane. Different dilutions, prepared by weight, were made for each sample type as a function of the density of the investigated sample. ABM and ABTM samples were diluted 5 and 10 times, respectively. Glycerol fractions, from HPLC, were collected according to retention time (tR = 16 min) and successively dried under a nitrogen stream. Just before the GC injection, glycerol samples were re-dissolved with ethanol to obtain a final glycerol concentration of about 3 g L−1. The standard glycerol solution was prepared by diluting a commercial sample to the same final concentration. 2.4. Equipment and instrumental settings Samples were weighed using a Gibertini E42S analytical balance (Gibertini Elettronica, Milan, Italy) with a sensitivity of ± 0.1 mg. HPLC quantitation and separation of glycerol, on the ABTM and ABM samples, were performed with a Beckman System Gold apparatus (Beckman Coulter, Milan, Italy), equipped with a 116 model isocratic pump, a 156 RI detector, a 406 model analogue interface module and a Rheodyne injector with a 100 μL loop. An Aminex HPX-87C column (300 × 7.8 mm), (Bio-Rad Laboratories S.r.L., Milan, Italy) packed with a styrene divinylbenzene resin, 8% crosslinked, thermostated at 75 °C, was used. The chromatographic separation was carried out with water as mobile phase with a flow rate of 0.6 mL min−1. Reproducibility of the instrumental method was checked by injecting a standard solution of glycerol twice a day, the corresponding standard deviation was associated to all data, SD = 0.1 g kg−1 (n = 20). The determination of the carbon stable isotopes ratio, 13C/12C, of the glycerol was carried out using a Thermo Finnigan Trace GC Ultra (Bremen, Germany) equipped with an Alltech Heliflex AT-WAX column (30 m × 0.25 mm × 0.20 μm; Analytical Columns, Croydon, UK). Helium was used as carrier gas and separation was performed at 1.1 mL min−1 flow rate. Carbon isotope ratio measurement was performed on-line using a GC Combustion Interface III connected to a Thermo Finnigan Delta Plus XP IRMS (Bremen, Germany). The gas chromatographic conditions were set as follows: injection temperature 270 °C, oven temperature program: initial temperature 120 °C for 2 min, then the temperature was increased with a rate of 15 °C min−1 to a final value of 240 °C, final hold time 5 min. The combustion oven was kept at 940 °C, while the reduction oven temperature was maintained at 650 °C. The gas stream containing the CO2 molecules, obtained from the polyalcohol oxidation, enters the ion source of the mass spectrometer where the CO2 molecules are ionized, accelerated and separated in a magnetic sector analyzer. CO2 ions, coming from the polyalcohol oxidation, were measured by means of Faraday cups centered on the 44, 45 and 46 mass-to-charge (m/z) values, respectively.
2.6. Statistical analysis Data elaboration was carried out with PLS toolbox 8.2 (Eigenvector Research Inc., Wenatchee, WA) in Matlab 9.0 environment (MathWorks Inc.). An alpha level of confidence of 0.05 was used for all statistical tests. 3. Results and discussion 3.1. Glycerol characterization An investigation was carried out relative to some commercial glycerol samples (A and B) obtained from different suppliers, and on several glycerol samples of different natural origin. Table 1 reports the δ13C values measured by GC-C-IRMS that can be useful for a better understanding of the general trend of δ13C of glycerol in samples of different origin. Considering the reported data, as expected, the plant or animal origin of the sample influence the δ13C values. In particular, glycerol from C3 plants such as: sunflower, olive, barley, and rice, gives δ13C values ranging from −26 to −30‰, while for C4 plants, δ13C values are more positive and close to −18‰. Values of δ13C evaluated on glycerol samples of animal origin cover a wide range, probably Table 1 δ13C (0/00) data for glycerol samples of different varietal origin and brand, the corresponding standard deviation is 0.2 0/00. glycerol commercial A commercial B butter lard sunflower oil olive oil S. cerevisiae malt barley S. cerevisiae malt cider S. bayanus malt rice S. cerevisiae malt maize S. bayanus malt maize
2.5. Calibration and isotopic calculation At the beginning of each run, three pulses of CO2 reference gas were admitted into the inlet system for about 20 s. The constant flow rate during this period gives these peaks a flat-top appearance. A level of CO2 corresponding to 3–5 V at m/z 44 was used to calibrate the system. The 13C/12C abundance ratio was expressed as δ13C values calibrated against the international standard Vienna Pee Dee Belemnite (VPDB) (Fry, 2006). The delta notation is defined as: 35
carbon fixation pathway
δ13C
C3 C3 C3 C3 C3 C4 C4
−24.2 −28.6 −30.4 −17.8 −28.1 −30.4 −26.5 −28.9 −29.3 −17.6 −18.8
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distribution, with no significant differences among the years as confirmed by the ANOVA result (F(3,88) = 0.48, p-value = 0.70). However, different considerations can be emphasized by considering the δ13C data reported in Fig. 2 and ANOVA data analysis reported in Table 2a. In fact, based on the plot distribution of the experimental values, it is possible to highlight a decreasing trend of the ABM means data over the years. Moreover, the mean of the ABM2008 distribution, δ13C = −27.95‰, is significantly higher with respect to the values obtained for the ABM2009, δ13C = −29.32‰, ABM2010, δ13C = −29.91‰ and ABM2011, δ13C = −30.4‰, as confirmed by the ANOVA results. Considering that the glycerol in ABM mainly comes from the wine vinegar fraction added to the product and that no limitation on the geographical origin of this raw material is imposed, the observed variability of the data is probably in accordance with a nonspecific geographical origin of the wine vinegar, namely wine vinegars coming from different countries with different climatic conditions (inter-variability), in addition to climatic changes between years (intravariability) (Gaudillere et al., 2002; Martin et al., 1999). Therefore, to better characterize the ABM product a long-term investigation and the creation of a dedicated database are suggested.
depending on the diet of the animal and the fractionation mechanisms that take place in the animal’s metabolism. As an example, the δ13C of lard is close to that of maize malt. These experimental data agree well with previously reported results (Fronza et al., 1998). Moreover, glycerol from maize malt produced by S. cerevisiae shows a higher δ13C value than that produced by S. bayanus, supporting evidence that different yeast strains may influence carbon isotopic composition of glycerol, as found for wine glycerol by Calderone et al. (2004). As far as δ13C values determined on commercial samples of glycerol (unknown origin) are concerned, these lay inside the variability range of animal and plant values. From a first glimpse of the data of Table 1 it is evident that several factors affect the δ13C values and, as a consequence of these overlaps, the possibility to differentiate sample origin/varietal/exogenous addition or provenance is rather difficult only based on this variable alone. 3.2. ABM samples To better understand the discriminating potential of glycerol δ13C, several ABM vinegars, coming from different producers in the province of Modena (Italy) and sampled in different production years from 2008 to 2011, were investigated. The glycerol concentrations and δ13C results are reported, as box and whiskers plots, in Figs. 1 and 2, respectively. Owing to the peculiarities of the ABM production process, the concentration of glycerol in the commercial product depends on the wine vinegar added to the concentrated must. In fact, as expected, the average glycerol content in ABM varied from 1.5 to 8 g kg−1 and was lower than in ABTM where values ranged from 3.8 to 20 g kg−1 or more. This evidence is coherent with the assumption that the amount of glycerol in the concentrated must is almost negligible, since no fermentation processes occur during its production. However, the level of the polyalcohol may be higher in products obtained from juices coming from mould-infected grapes (Botrytis cinerea) where glycerol is produced in the grape berries during the ripening period (Lorenzini et al., 2012). Over the investigated years, the ABM samples show a constant trend of glycerol content both as concentration range and as interquartile
3.3. ABTM samples and cask series The analytical procedure was also applied to 20 ABTM samples and the experimental data are reported in graphical form in Figs. 1 and 2. Concerning the glycerol content, Fig. 1, the ABTM samples show a quite wide distribution that ranges from 8.7 to 22 g kg−1. Glycerol concentration ranges, typically, from 2 to 20 g kg−1 in wines and to 50 g kg−1 in aged traditional balsamic vinegars (Calderone et al., 2004; Turtura and Grasselli, 2003). The concentration values are in agreement with literature data and with the peculiarities of the ABTM chain process (alcoholic fermentation followed by acetic bio-oxidation and concentration effect during the long ageing period due to the topping up procedure and the loss of water). At the same time the glycerol content, evaluated along the investigated cask series, agrees well with the concentration range measured for the marketable ABTM. As a matter of fact, a direct comparison of the ABTM data with the BatA to BatC distributions is not possible since the casks contain an evolving
Fig. 1. box and whiskers plot of the glycerol concentration, g kg−1, measured for ABM, years 2008–2011, marketable ABTM and cask series BatA to BatC. The horizontal line inside the box represents the median value of the data; the dot represents the mean value. Circles represent the outliers.
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Fig. 2. box and whiskers plot of the δ13C of glycerol measured for ABM, years 2008–2011, marketable ABTM and cask series BatA to BatC. The horizontal line inside the box represents the median value of the data; the dot represents the mean value. Circles represent the outliers.
considered all of endogenous origin. The latter concerns BatC, where the average δ13C (−26.6 ± 1.4‰) is higher than in ABTM. The ANOVA (Table 2b) confirms that there is a meaningful difference between groups and, considering the plot of Fig. 2, it is clear to see that this is due to BatC samples distributions. Taking into account that the production rule for ABTM limits the use of raw materials, in terms of geographical origin, to grapes coming from the province of Modena and considering that seasonal variation is not so pronounced within the production area, it is quite surprising that BatC glycerol δ13C variability lay outside the variability range of the marketable ABTM. The most plausible reason for this behavior could be deviation from the traditional preparation of this cask series in terms of raw material, such as the use of mixtures of concentrated musts and wine vinegars of different geographical origins and/or varietals. This consideration is also supported by the evidence that the upper interquartile limit lies outside the above mentioned interval for vine plants, highlighting the hypothesis that some glycerol is derived from sugars of exogenous origin (Hattori et al., 2010). Moreover, considering the cut off value δ13Cethanol of −22‰ and the minimum δ13Cethanol of −30‰ observed for biotic C3 carbon fixation mechanism for vine plants (Christoph et al., 2015 and references herein reported; Smith and Epstein, 1971), and that glycerol is more depleted in δ13C as previously reported, the above interval for vine plants should be equivalent to a δ13C scale, measured for glycerol, that ranges from −25/–26 to −33/–34‰. As a consequence, ABTM data are coherent with grapes being used as the raw materials.
Table 2 One-way ANOVA to test for significant differences in ABM data (a) and ABTM data (b). a) source of variation
sum of squares
degree of freedom
F
F crit (α = 0.05)
between groups within groups
60.7126 65.9632
3 88
26.9984
2.70819
b) source of variation
sum of squares
degree of freedom
F
F crit (α = 0.05)
between groups within groups
56.09658 29.27586
3 41
26.18721
2.832747
product and, on the basis of the topping up procedure, only the glycerol content of the vinegar of the oldest barrels could be compared with the ABTM data (Cocchi et al., 2002). As regard the carbon isotope ratio, Fig. 2 reports the δ13C data evaluated on both the ABTM commercial products and the samples coming from the three cask series (BatA, BatB and BatC). Now, starting from the consideration that the δ13C value depends on the plant type and on the provenance of the grapes, it is possible to highlight that, for the ABTM commercial products, all the values are more or less distributed close to the average δ13C = −29.4 ± 0.5‰. Owing to the absence, to our knowledge, of other experimental data evaluated on the same matrix and considering the present numbers as face values, it is reasonably possible to assess that the glycerol, separated and measured in the ABTM, has the same natural origin. On the other hand, taking into account data obtained from the analysis of vinegars from three different cask series, two main conclusions can be drawn. The former is that BatA (δ13C = −29.3 ± 0.9‰) and BatB (δ13C = −29.9 ± 0.8‰) show average values coherent with the one determined on marketable ABTM. Hence there is no evidence of a particular trend inside the two series, confirming that the glycerol is formed during the alcoholic fermentation of grape sugars and it may be
4. Conclusion The use of the GC-C-IRMS approach, for the determination of δ13C of glycerol in balsamic vinegars of Modena, was a satisfactory technique for a deeper knowledge of this complex matrix. This preliminary study focuses on the evaluation of the potentialities of the isotopic methodology in discriminating glycerol of different sources and products with different chain processes, such as ABTM and ABM. In particular, glycerol concentration and δ13C results were coherent with grape-derived products for almost all of the investigated samples. Moreover, ABTM, due to its peculiar manufacturing process, shows a 37
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restricted δ13C variation for glycerol compared to the homologous industrial ABM. Nevertheless, taking into account the limited number of samples under investigation and despite the positive evidence obtained in the present study, the use of only one molecular marker was not sufficient to assess the authenticity of balsamic vinegars. In fact, in some cases the huge variability of the data may not allow discrimination of similar products or highlight fraudulent procedures. In conclusion, as seasonal and regional factors affect the level of isotope, data from other authentic balsamic vinegars have to be considered over a long-term investigation with the implementation of a dedicated data bank. Furthermore, monitoring more isotopic patterns in different molecules, e.g., acetic acid, in the case of vinegar or similar products, might enhance the diagnostic potential of the isotopic approach.
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