MW ROUND TABLE #1 – Identity, typicality, traceability

Genetic traceability of ‘Nebbiolo’ musts and wines by single nucleotide polymorphism (SNP) genotyping assays

Giorgio GAMBINO, Paolo BOCCACCI, Anna SCHNEIDER – Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), Italy.
Walter CHITARRA, Council for Agricultural Research and Economics, Viticultural and Enology Research Centre (CREA-VE), Italy.
Luca ROLLE, Department of Agricultural, Forest and Food Sciences, University of Torino, Italy.
Email contact: giorgio.gambino[@]ipsp.cnr.it

AIM: ‘Nebbiolo’ (Vitis vinifera L.) is one of the most ancient and prestigious Italian grape cultivars. It is renowned for its use in producing monovarietal high-quality red wines, such Barolo and Barbaresco. Wine quality and value can be heavily modified if cultivars other than those allowed are employed. The fight against fraud to safeguard high-quality productions requires an effective varietal identification system applicable in musts and wines.

METHODS: Single-nucleotide polymorphisms (SNPs) are considered the newest type of molecular marker for grapevine identification. We developed and investigated the efficiency of SNP TaqMan® assays in the varietal authentication of ‘Nebbiolo’ musts and wines. ‘Nebbiolo’-specific SNPs were identified starting from available databases and 260 genotypes analysed by Vitis18kSNP array.

RESULTS: Only two markers (SNP_15082 and SNP_14783) were sufficient to distinguish ‘Nebbiolo’ from more than 1,100 genotypes. In experimental vinifications, these SNP TaqMan® assays correctly identified ‘Nebbiolo’ in all wine-making steps, including wines 1 year after bottling. The high sensitivity of the assays allowed identifying, for the first time, mixtures of 1% in musts at the end of maceration, blends of 10% in musts at the end of malolactic fermentation and wines contamination of 10–20% with non-‘Nebbiolo’ genotypes. In commercial wines, the amplification efficiency of these SNPs was partially limited by the low amount of grapevine DNA and the presence of PCR inhibitors in DNA extracts. However, at least one SNP amplified correctly in all the commercial wines tested.

CONCLUSIONS: The TaqMan® genotyping assay is a rapid, highly sensitive and specific methodology with remarkable potential for varietal identification in wines.

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NMR profiling of grape musts from some Italian regions

Pavel Solovyev¹, Matteo Perini¹, Pietro Franceschi¹, Luana Bontempo¹, Federica Camina^2
1Fondazione Edmund Mach (FEM), Italy
²University of Trento, Italy
Email contact: pavel.solovyev[@]fmach.it

With wine fraud, being a widespread problem [1], the need for more sophisticated and precise analytical methods of its detection remains ever persistent. Nuclear magnetic resonance (NMR) spectroscopy has been widely used for analysis of wine in recent years [2,3], but wine musts were much less studied; in fact, only one paper dealt with the NMR spectra of actual musts [4]. Difficulties arise mostly because grape musts are “live” objects, which undergo rapid fermentation at room temperature, if not inhibited either by freezing or chemical preservative; but even such measures are not sufficient to halt it completely [5].

We have investigated over 300 samples of grape must from 17 of 20 different Italian regions using ¹H NMR spectroscopy with water signal suppression, postprocessing in the MatLab software with dynamic alignment [6] and optimized binning [7] to alleviate the effect of fermentation on the chemical shifts of mobile protons. After that, multivariate statistics was performed with techniques such as PCA, PLS-DA and OPLS-DA with respect to various group parameters such as regions, vitivinicultural zones, harvest periods and grape varieties. Advantages and drawbacks of each method were addressed.

References:
1. Lin S. et al. Food Fraud. Academic Press; 2021. 233-247.
2. Sobolev A.P. et al. Trends Food Sci Technol. 2019; 91: 347-353.
3. Solovyev P.A. et al. Compr Rev Food Sci Food Saf. 2021; 20, 2: 2040-2062.
4. López-Rituerto E. et al. J Agric Food Chem. 2012; 60: 3452-3461.
5. Flamini R. J Food Process Preserv. 2007; 31: 345-355.
6. Savorani F. et al. J Magn Reson. 2010; 202: 190-202
7. Sousa S.A.A. et al. Chemometrics Intellig Lab Syst. 2013;122: 93-102

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Neural Networks and FT-IR spectroscopy for the discrimination of single varietal and blended wines. A preliminary study

Marianthi BASALEKOU, Anna Georgoulaki, Anna STEFOU – University of West Attica, Greece
Christos PAPPAS, Petros TARANTILIS – Agricultural University of Athens, Greece
Email contact: mbasalekou[@]uniwa.gr

Blending wines from different grape varieties is often used in order to increase wine complexity and balance. Due to their popularity, several types of blends such as the Bordeaux blend, are protected by PDO legislation. In the case of monovarietal wines blending is forbidden, however there is no method to authenticate their status, and for this reason adulteration can are difficult to identify. Fourier Transform Infrared Spectroscopy (FT-IR) has proven successful for the discrimination of wines based on several parameters such as geographical origin and type of aging, while the use of Neural Networks is now used more often for the development of prediction models. FT-IR spectroscopy coupled with Neural Networks have been used to develop a prediction model for the discrimination of single varietal and blended wines. Generalized RSquare for the training set was 0,9011 and 0,689 for the validation set, while the -Loglikelihood was 3,918 for the training and 0,111 for the validation set. The misclassified rate was 0,03 for the training set and 0,11 for the validation set, showing very good potential for the use of IR spectroscopy for the authentication of single varietal and blended wines.

Reference:
Basalekou, Marianthi, Christos Pappas, Petros A. Tarantilis, and Stamatina Kallithraka. “Wine Authenticity and Traceability with the Use of FT-IR.” Beverages 6, no. 2 (2020): 30. https://doi.org/10.3390/beverages6020030

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Insights from Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) and chemometrics applied to the quick discrimination of grapevine varieties

Thomas BAERENZUNG dit BARON, PPGV, INP-PURPAN, University of Toulouse.
Alban JACQUES, PPGV, INP-PURPAN, University of Toulouse
Olivier GEFFROY, PPGV, INP-PURPAN, University of Toulouse
Valérie SIMON, LCA, INP-ENSIACET, University of Toulouse
Olivier YOBRÉGAT, IFV Sud-Ouest
Email contact: thomas.baerenzung-dit-baron[@]purpan.fr

Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) is an innovative analytical method based on soft chemical ionization to analyze thecomposition in volatile compounds of a gas phase.

In this research, we propose a quick way to access the aromatic potential of grape varieties through a scan of their volatilome by SIFT-MS and chemometrics approaches.

During 3 sampling campaigns carried out in September 2020, we collected berries from 21 grapes varieties planted in a germplasm collection. For each variety, three replicate samples of 50g were gently crushed and put in 1L Schott bottles that were directly connected to a SIFT-MS equipment to analyse the headspace. Analytes  injected in the SIFT-MS were ionized with 3 different reagent ions (H₃O⁺, O₂^+. and NO⁺) to generate increased molecular fragmentation data (2).

M/z data/ratios were first analysed with XlStats software (Addinsoft, Paris, France) using a one-way ANOVA treatment to determine the ions that enabled to discriminate the grape varieties. Then based on these discriminating ions, Principal Component Analysis (PCAs) were constructed and Hierarchical Clustering Analysis (HCA) ensued to create similarity groups. Finally, an ANOVA treatment was conducted to determine significant differencies in ions abondances between groups (1).

For each homogenous group, a cultivar was selected to perform Headspace-Solid Phase Microextraction (HS-SPME) followed  by Gas Chromatography-Mass Spectrometry (GC-MS) analyzes to connect SIFT-MS data to the composition in volatile compounds (3).

Grape varieties were easily distinguishable based only on their SIFT-MS volatilome scan. The technique was able to distinguish high and low aroma compounds producers, and to organise grape varieties by similarity.

We proved that SIFT-MS is a really quick and interesting tool with potential application in various fieds of viticulture such as phenotyping of grape varieties based on their volatile composition or studying of the impact of viticultural practices on the grape aroma composition using an easy to implement untargeted approach.

References:

1. Cappellin, L. et al. PTR-ToF-MS and data mining methods: a new tool for fruit metabolomics. Metabolomics 8, 761–770 (2012).
2. Spanel, P. et al. Quantification of volatile compounds in the headspace of aqueous liquids using selected ion flow tube mass spectrometry. Rapid Commun. Mass Spectrom. 16, 2148–2153 (2002).
3. Ozcan-Sinir, G. et al. Detection of adulteration in extra virgin olive oil by selected ion flow tube mass spectrometry (SIFT-MS) and chemometrics. Food Control 118, 107433 (2020).