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A novel 2D-NMR approach for the classification of balsamic vinegars of Modena Riccardo Graziosi, Davide Bertelli, Lucia Marchetti, Giulia Papotti, Maria Cecilia Rossi, and Maria Plessi J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 09 Jun 2017 Downloaded from http://pubs.acs.org on June 11, 2017
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Journal of Agricultural and Food Chemistry
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A novel 2D-NMR approach for the classification of balsamic vinegars of Modena
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(Riccardo Graziosia, Davide Bertellia*, Lucia Marchettia, Giulia Papottia, Maria Cecilia Rossib, Maria Plessia)
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a
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Modena, Italy
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b
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via Campi 213⁄A, 41125 Modena, Italy
Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia, via Campi 103, 41125
Centro Interdipartimentale Grandi Strumenti, Università degli Studi di Modena e Reggio Emilia,
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∗Corresponding author: Dr. Davide Bertelli, Dipartimento di Scienze della Vita, Università di
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Modena e Reggio Emilia, via Campi 103, 41125 Modena, Italy.
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Tel.: +39 059 2058561;
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E-mail address:
[email protected] (D.Bertelli).
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Abstract
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The aim of this work is to evaluate the possibility of using 2D-NMR for the construction of
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classification models for balsamic vinegars of Modena. The goal was to obtain an indirect indicator
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of authenticity and a quality control tool. The spectral data were analysed by chemometric methods,
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aiming to discriminate the samples in relation to their origin. Application of general discriminant
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analysis (GDA) revealed a good discrimination, the two obtained models explained 83.9% and
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97.3% of the total variance with a predictive capacity of 98.6% and 98.4 % respectively. The
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signals of 5-HMF, β-glucose, 2,3-butanediol, 6-acetyl glucose, and different aliphatic signals of
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sugars were the most significant variables. These results are very promising for giving an important
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contribution in quality control and characterization of such very valuable foods.
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Keywords:
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NMR, Two-dimensional NMR, Traditional Balsamic Vinegar of Modena, chemometrics
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INTRODUCTION
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Balsamic vinegar of Modena (BVM) and traditional balsamic vinegar of Modena (TBVM) are two
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important and valuable Italian foods appreciated all over the world. The productions of these
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vinegars are regulated by European Union, this guarantee makes them more valued especially by
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consumers. Considering its peculiar production procedure and the limited geographical area of
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origin, TBVM had been granted as Protected Designation of Origin (PDO) product (EC Council
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Regulation No. 813/2000). BVM has achieved the registration with Protected Geographical
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Indication (PGI) status (Reg. CE n. 583/2009 July 3th, 2009).
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The annual production of BVM is over 90 million liters, the 90% of which is exported in over 120
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countries with a money turnover around 700 million euros, thanks to this BVM is one of the most
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important Italian protected food specialties. TBVM is a more valuable traditional product and its
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production levels are significantly lower than BVM. In the last years a high increase, especially on
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foreign commerce, of imitative products that try to take advantage from the brand image and
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sounding of TBVM and BVM to commercialize similar but not original and cheaper poor quality
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seasonings has occurred. The economic damage was estimated at around 60 million euros a year.
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(Data obtained from Consortium for protection of balsamic vinegar of Modena). The principal
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difference between balsamic vinegars and other similar seasonings is that the former are produced
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starting from cooked and/or concentrated grape must while the latter are obtained normally from
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alcoholic beverages (i.e. wine). Although BVM and TBVM share some characteristics, but many
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differences justify the distinction between the two recognized typologies. The principal differences
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between TBVM and BVM concern the production method and the ageing process. According to the
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tradition TBVM is aged in barrels made with different kind of woods (i.e. oak, chestnut, juniper,
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cherry, mulberry, robinia) and is commercialized as two different products according to the ageing 3
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period: standard product with ageing that lasts from 12 to 25 years and “extravecchio” (extra old)
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aged more than 25 years, this last definition can be included in the label. BVM is a cheaper
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industrial product, with a shorter maturation period, mandatorily conducted in wooden cask from 2
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months up to 3 years. The ageing period is not the unique variable that differentiates these typical
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Italian vinegars, but also the type of ageing has to be taken into account. The chemical, physical and
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organoleptic characteristics of these vinegars strongly depend on the starting material. TBVM is
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obtained from cooked and concentrated grape must; the regulation allows the inoculation of acetic
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bacteria, while any extra additive is forbidden. Instead the regulation for BVM allows the use of:
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cooked and concentrated must, wine vinegar (10% v/v minimum) and caramel (2% v/v maximum)
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for color correction (Decreto Ministeriale, 3 Dicembre 1965). All these variables contribute to
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differentiate one vinegar from another.
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Different works published report studies that aim to study chemical-physical characteristics of
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BVMs and TBVMs, to establish a relationship with their production procedure, to characterize
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these products and protect them from commercial fraud.1-8 Usually assessments regarding the
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quality and the authenticity of balsamic vinegars are performed using organoleptic analysis and the
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measure of a considerable number of ordinary chemical-physical determinations e.g., dry matter,
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density, total and volatile acid content. The results provided by these analyses have been
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occasionally used to classify balsamic vinegars, moreover they are often used, in association with
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other analytical data (i.e. mid-infrared spectroscopy), for the qualitative assessment and the
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prediction of sensory qualities.9-11
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Different analytical methods have been employed for the characterization and for the age
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determination of these premium quality vinegars. Several authors used solid-phase extraction
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(SPE), dynamic headspace or headspace solid-phase microextraction (HS-SPME) coupled with gas 4
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chromatography to study the volatile fraction. 1,12-14 Theobald et al. used a simple High-
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Performance Liquid Chromatography (HPLC) method with minimum sample preparation to
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investigate the 5-hydroxymethylfurfural (5-HMF) content in different vinegar samples, traditional
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balsamic vinegars among them, in order to verify its usability as mean to assess vinegars age.15
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Although different approaches are reachable in literature none of them appear to be really
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conclusive about the classification and authentication issues for balsamic vinegars from Modena.
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For this reason some years ago we decided to investigate the possibility to use NMR to deal with
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the question.
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During the last years, High-Resolution Nuclear Magnetic Resonance spectroscopy (HR-NMR) has
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been proved to be a very promising method for food quality assessment.16-21 It is a well suited
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technique that allows to analyze samples with a very limited sample preparation, avoiding in this
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way the artifacts formation, moreover NMR is not a destructive analysis. In particular one-
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dimensional technique, characterized by simple acquisition procedure and very competitive time
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consumption has demonstrated effectiveness in a large range of applications. This work describes
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the use of two-dimensional NMR (2D NMR) spectroscopy in order to obtain a fingerprint of several
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BVMs and TBVMs, useful for their characterization and discrimination. Among the numerous
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scientific works dealing with food characterization and NMR, available in the scientific literature,
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only a limited number of articles reports the use of this methodology applied to TBVM and BVM
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study.22-25 These studies preferentially employed one-dimensional NMR (1D NMR) owing also to
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the difficulty of using 2D spectra, especially when coupled with statistical analyses. In our past
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efforts we used 1D NMR coupled with multivariate data analysis to characterize BVM and
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TBVM.26 We also used compositional and functional data of the same type of vinegars joined with
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H NMR spectroscopy to obtain multivariate classification models.27 In 2D NMR very cluttered 5
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spectra are often obtained, especially in the case of complex mixtures as balsamic vinegars are.
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Usually two-dimensional NMR normally requires a longer acquisition time compared to one-
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dimensional, however it often allows to gain an higher resolution in presence of overlapped signals.
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This fact justifies our attempt to use two-dimensional NMR in the study of complex natural
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matrices as balsamic vinegars are. These spectra require powerful data analysis methods to obtain
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statistically significant results. To overcome this limitation, we used Matlab® environment to read
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and easily manipulate 2D NMR spectra allowing the extraction of spectral information exploitable
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for the following statistical analyses. The goal was to obtain an indirect indicator of authenticity and
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a quality control tool. The data resulting from 2D NMR spectra were analysed by chemometric
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methods, aiming to discriminate the samples in relation of the type of vinegar. This application gave
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an important contribution in quality control and characterization of such very valuable foods, with
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limited sample manipulation. Furthermore this study aimed to demonstrate the real usefulness of
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two-dimensional NMR spectra obtained from the analysis of complex food mixtures for the
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construction of classification models.
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MATERIALS AND METHODS
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Materials and sample preparation
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A total of 64 samples of both TBVM and BVM have been analyzed. Among them, 22 were “extra
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old” PDO (>25 years of ageing) TBVM, 15 were standard PDO (>12 and
