Development of a qPCR method for classification of botrytized grape berries originated from Tokaj wine region.

One of the best-known Hungarian products on world wine market is Aszú, which belongs to the family of Tokaj wine specialties and is made from aszú berries. An important condition for the formation of aszú berries is the noble rot of technologically mature grapes, which is caused by Botrytis cinerea. At the same time botrytized sweet wines are produced not only in Hungary, but in many locations of wine-producing areas of Europe as well as in certain wine growing regions of other continents. The determination of botrytization is mostly based on sensory evaluations, which is a highly subjective procedure and largely depends on the training and experience of the evaluator. Currently, the classification of aszú berries (class I and class II) is based only on visual inspection and determination of sugar content. Based on these facts the primary goal of our work was to develop a qPCR assay capable for objective rating and classification of aszú berries. The developed qPCR is highly specific and sensitive as can clearly distinguish between B. cinerea and other filamentous fungi and yeast species occur on grapes. Moreover, it is suitable for categorizing berries colonized by B. cinerea to varying degrees. Thus, the developed qPCR method can be a useful technique for classification of the grape berries into four quality groups: healthy, semi-shrivelled, Aszú Class II and Aszú Class I.

A digital image smartphone-based approach to Slovak Tokaj wine authentication chemometric assisted.

The authentication of Slovak wines in comparison to other similar wines from various geographical regions, namely Hungary, France, Austria, and Ukraine, was conducted using the OC-PLS, DD-SIMCA, and PLS-DM models, all of them operating in rigorous way. The study involved 63 samples, of which 41 originated from Slovakia, covering diverse wine types such as varietal wines, cuvée selections (different "putnový"), and essence. To capture digital images under controlled conditions, a custom-made cardboard box with white inner surfaces was devised and equipped with a smartphone. During the training phase, sensitivities of 96%, 100%, and 96% were attained for OC-PLS, DD-SIMCA, and PLS-DM, respectively. In the subsequent stages of validation and testing for DD-SIMCA and PLS-DM, the proposed methods displayed optimal efficiency, achieving both sensitivity and specificity rates of 100%. However, such results were not achieved in the case of OC-PLS, which exhibited efficiency levels of 90% in validation and 80% in testing.

A non-target geographical origin screening of botrytized wines through comprehensive two-dimensional gas chromatography coupled with high-resolution mass spectrometry.

One of the most effective methods for gaining insight into the composition of trace-level volatile organic characteristics of wine products is through the use of a comprehensive two-dimensional gas chromatography-high resolution mass spectrometry (GC × GC-HRMS) technique. The vast amount of data generated by this method, however, can often be overwhelming requiring exhaustive and time-consuming analysis to identify significant statistical characteristics. The use of advanced chemometric software can achieve the same or even higher efficiency. This study aimed to identify differences based on geographical locations by analyzing the volatile organic compounds in the composition of botrytized wines from Slovakia, Hungary, France, and Austria. The volatile organic compounds were extracted by solid-phase microextraction and analyzed using GC × GC-HRMS. The data obtained from the analysis underwent Fisher-ratio (F-ratio) tile-based analysis to identify statistically significant differences. Principal component analysis demonstrated a significant distinction between wine samples based on geographical location, using only 10 statistically significant features with the highest F-ratio. In the samples, the following compounds were analyzed: methyl-octadecanoate, 2-cyanophenyl-ß-phenylpropionate, α-ionone, n-octanoic acid, 1,2-dihydro-1,1,6-trimethyl-naphthalene, methyl-hexadecanoate, ethyl-pentadecanoate, ethyl-decanoate, and γ-nonalactone. These, all play an important role in cluster pattern observed on principal component analysis results. Additionally, hierarchical cluster analysis confirmed this.

Enhanced multi-stir bar sorptive extraction for wine analysis: Alteration in headspace mode.

Multi-stir bar sorptive extraction (multi-SBSE) represents a viable alternative for recent trends in sample preparation based on a combination of extraction techniques. In this case, increased amount of sorbent and its extended polarity range could advance quality of experimental data obtained in foodomics or metabolomics investigations. With this in mind, it was developed multi-SBSE procedure suitable for authentication of botrytized wine produced in different countries of Tokaj wine region. A design of headspace mode of multi-SBSE was modified to provide additional agitation of a stir bar. An expanded profile of wine samples was obtained with the application of EG-Silicone and PDMS coated stir bars in headspace and direction immersion mode, respectively. Multivariate optimization based on central composite design was selected to determine the influence of various experimental parameters, including extraction temperature enhancing headspace extraction. In our case, proper description of the optimization results required application of a third-order polynomial model, which highlighted dominant influence of salt addition for extraction in both modes. Due to a large number of extracted compounds, comprehensive two-dimensional gas chromatography (GC × GC) was used for assessment of wine samples. Such approach allows reveleaing the presence of sulphur containing compounds, diols, ketone derivatives and methoxybenzenes linking a specific geographical origin. At the same time, the results obtained for compounds common for all the samples were processed with principal component analysis (PCA). Considerable progress for discrimination of the botrytized wines was mainly achieved with combined data from EG-Silicone and PDMS extraction.

Optimization of enantiomer separation in flow-modulated comprehensive two-dimensional gas chromatography by response surface methodology coupled to artificial neural networks: Wine analysis case study.

In spite of extensive applications of flow modulated comprehensive two-dimensional gas chromatography (FM-GG × GC) in different research areas, its application in the field of chiral separation is very limited. From a practical point of view, the establishment of experimental parameters for enantiomer separations is possibly more demanding in this case. Since the carrier gas flows in both dimensions, it affects not only the separation parameters, but also the fill/flush volumes of the modulator and its working efficiency. In this context, a multivariate design of experiment was applied to find the optimum experimental parameters of a reversed fill/flush (RFF) modulator for enantiomer separation of organic compounds present in botrytized wine samples. The results were described both with response surface methodology and artificial neural networks (ANN). The enantiomeric composition of chiral compounds present in the botrytized wines was used to identify their geographical origin, by principal component analysis (PCA). In addition, the developed one-class partial least squares (OC-PLS) model enabled recognition of the wine samples from the Tokaj wine region with 93% effectiveness in the presence of other samples.

Redox and Hormonal Changes in the Transcriptome of Grape (Vitis vinifera) Berries during Natural Noble Rot Development.

Noble rot is a favorable form of the interaction between grape (Vitis spp.) berries and the phytopathogenic fungus Botrytis cinerea. The transcriptome pattern of grapevine cells subject to natural noble rot development in the historic Hungarian Tokaj wine region has not been previously published. Furmint, a traditional white Tokaj variety suited to develop great quality noble rot was used in the experiments. Exploring a subset of the Furmint transcriptome redox and hormonal changes distinguishing between noble rot and bunch rot was revealed. Noble rot is defined by an early spike in abscisic acid (ABA) accumulation and a pronounced remodeling of ABA-related gene expression. Transcription of glutathione S-transferase isoforms is uniquely upregulated, whereas gene expression of some sectors of the antioxidative apparatus (e.g., catalases, carotenoid biosynthesis) is downregulated. These mRNA responses are lacking in berries exposed to bunch rot. Our results help to explain molecular details behind the fine and dynamic balance between noble rot and bunch rot development.

Handling multiblock data in wine authenticity by sequentially orthogonalized one class partial least squares.

New approach to deal with food authentication by modelling methods based on data recorded from different sources is proposed and called OC-PLS, combines an orthogonalization step between the different data sets to eliminate redundant information followed by definition of an acceptance area for a target class by OC-PLS. The proposed method was evaluated in two case studies. The first study used a controlled scenario with simulated data. In the second case study, the approach was applied using UV-VIS and IR data, in order to differentiate Slovak Tokaj Selection wines of high quality from other lower market value wines from the Slovak Tokaj wine region. In both cases, better results were reached than when individual blocks of data were achieved. The proposed method proved to be effective in properly exploring common and distinct information in each data block. The best compromise between sensitivity and selectivity in the prediction step was achieved.

Multivariate optimization of dual-sorbent dynamic headspace extraction of volatiles in wine analysis.

The main critical point of newly developed miniaturized sample preparation techniques is a limited extraction capacity. Dynamic headspace extraction offers increased volume of sorbent which is commonly used in environmental analysis. Application of two sorbents (Carbopack B/Carbopack X and Tenax® TA) at different extraction temperatures allows enhancing a range of volatile organic compounds available for analysis. Such approach was applied in our research for quantification of volatile organic compounds in botrytized wines with gas chromatography. The central composite design was included to analysis simultaneous effects of incubation time, incubation temperature, purge volume and purge flow. In attempt to properly assess results, the data evaluation involved Pareto charts, surface response methodology and principal component analysis. Multivariate experimental design revealed statistical significance of purge volume and quadratic terms of incubation time and temperature, for response of volatiles. The quantification method with 0.2-2.0 µg/L LOD and 0.5-5.0 µg/L LOQ values, was developed under simultaneously optimized experimental conditions such as a 54 °C incubation temperature, a 20.18 min incubation time, a 344.3 mL purge volume and a 16.0 mL/min purge flow. The increased levels of linalool oxide, ethyl phenylacetate, γ-hexalactone and α-terpineol were observed in the samples, that correlated with botrytized wine technology.

Classification of Botrytized Wines Based on Producing Technology Using Flow-Modulated Comprehensive Two-Dimensional Gas Chromatography.

The enantiomeric ratio of chiral compounds is known as a useful tool to estimate wine quality as well as observe an influence of wine-producing technology. The incorporation of flow-modulated comprehensive two-dimensional gas chromatography in this type of analysis provides a possibility to improve the quality of results due to the enhancement of separation capacity and resolution. In this study, flow-modulated comprehensive two-dimensional gas chromatography was incorporated in enantioselective analysis to determine the influence of winemaking technology on specific features of botrytized wines. The samples included Tokaj essences (high-sugar wines), Tokaj botrytized wines and varietal wines (Furmint, Muscat Lunel, Lipovina) and wines maturated on grape peels. The obtained data was processed with hierarchic cluster analysis to reveal variations in composition and assess classification ability for botrytized wines. A significant difference between the samples was observed for the enantiomeric distribution of ethyl lactate and presence of monoterpene alcohols. The varietal wines were successfully separated from the other types, which showed more similar results and could be divided with additional parameters. We observed a correlation between the botrytized wines and the varietal wines fermented with grape skins. As to the essences produced from juice of botrytized grapes, the results were quite similar to those of the botrytized wines, even though monoterpenes were not detected in the extracts.

Slovak Tokaj wines classification with respect to geographical origin by means of one class approaches.

Tokaj wines could be produced only in so called Tokaj/Tokay wine region that falls within two countries Slovakia and Hungary. Thus, wines bearing Tokaj appellation must be produced only in Hungary and Slovakia by traditional process. Unfortunately, some counterfeit wines from neighbour region in Ukraine could be found in market. The aim of this work is to explore a simple UV-VIS spectrum to recognise true Tokaj/Tokay wines from counterfeits and try to differentiate wines based on their country of origin. This type of question can be duly answered using one class classification approach. Two different approaches, Data Driven Soft Independent Modelling of Class Analogy - DD-SIMCA and One-Class Partial Least Squares - OC-PLS were tested and evaluated for this purpose. In both cases, rigorous way models were built and optimized using only samples of the target class. A set of external samples containing samples from target class and non-target were used to validate the models ability to recognize Slovak samples and reject non-Slovak samples. Model based on DD-SIMCA showed better performance (97% correct rating) compared to OC-PLS models (80% correct rating). Comparing both approaches in terms of sensitivity and specificity, both exhibit high sensitivity (low false negative rate: DD-SIMCA 95% and OC-PLS 100%), however the OC-PLS based model showed low specificity (40%) while DD-SIMCA showed high specificity (100%) rejecting all samples out of Slovak origin. Therefore, the results found in this study show that it is possible to successfully combine UV-VIS spectra and DD-SIMCA models to discriminate Tokaj wine samples of Slovak origin from others. Equally important is environmentally friendly (fast, simple, absence of solvents) classification method in line with green chemistry.

A fast and inexpensive approach to characterize Slovak Tokaj selection wines using infrared spectroscopy and chemometrics.

The Tokaj Selection wines (hungarian equivalent "Aszú") are typical noble sweet wines produced in Tokaj wine region that falls within two countries, Slovakia and Hungary. Taking into account the economic importance and uniqueness of these wines, in this work, a new, fast and inexpensive method that combines infrared spectroscopy and multivariate models for characterization Slovak Tokaj Selection wines was developed. At first, sample authentication via one class models (dd-SIMCA) considered Slovak Tokaj Selection wines as target class. The non-target sample was considered to be only a Tokaj sample of Slovak origin. The resulting model was able to properly recognize samples of the target class with high sensitivity and specificity. Subsequently, the putna index was predicted via PLS models. RMSEP equals 0.44; REPpred of 9.6 and R2 0.95 was found in prediction step.

Volatile composition and enantioselective analysis of chiral terpenoids in Tokaj varietal wines.

The present work compares VOC profiles of 37 Tokaj varietal wines produced from Furmint, Lipovina and Muskát zltý grape varieties determined by SPME-GC-MS. The identified VOCs underwent treatment using one-way ANOVA and PCA for determination of compounds capable to distinguish wines based on grapevine variety. The presence of high concentration of terpenoids was characteristic for Muskát zltý wines, while Furmint and Lipovina expressed quite high similarity, nevertheless Lipovina was characterized by higher relative concentration of 1,1,6-trimethyl-1,2-dihydronaphtalene. Enantioselective analysis of the dominant terpenoids (limonene, linalool, hotrienol and α-terpineol) was performed by heartcut-2D-GC: R form was dominant for limonene and linalool and S enantiomer for hotrienol and α-terpineol. Statistical analysis (ANOVA) confirmed R-linalool as the most significant compound responsible for the main differences between studied varietal wines. LDA identified both total linalool and R-linalool as the only significant (p ≤ 0.05) sorting keys to distinguish investigated varietal wines. Both models allow perfect distinguishing of Muskát zltý from Lipovina and Furmint (p ≤ 0.05) with posterior probability of re-identification 100% but low distinction of Lipovina and Furmint from each other. The probability of posterior re-identification of Lipovina raised from 76.9 to 84.6% and total probability from 86.5 to 89.2% when using dominant enantiomer concentration as sorting variable.

Vinification without Saccharomyces: Interacting Osmotolerant and "Spoilage" Yeast Communities in Fermenting and Ageing Botrytised High-Sugar Wines (Tokaj Essence).

The conversion of grape juice to wine starts with complex yeast communities consisting of strains that have colonised the harvested grape and/or reside in the winery environment. As the conditions in the fermenting juice gradually become inhibitory for most species, they are rapidly overgrown by the more adaptable Saccharomyces strains, which then complete the fermentation. However, there are environmental factors that even Saccharomyces cannot cope with. We show that when the sugar content is extremely high, osmotolerant yeasts, usually considered as "spoilage yeasts", ferment the must. The examination of the yeast biota of 22 botrytised Tokaj Essence wines of sugar concentrations ranging from 365 to 752 g∙L-1 identified the osmotolerant Zygosaccharomyces rouxii, Candida (Starmerella) lactis-condensi and Candida zemplinina (Starmerella bacillaris) as the dominating species. Ten additional species, mostly known as osmotolerant spoilage yeasts or biofilm-producing yeasts, were detected as minor components of the populations. The high phenotypical and molecular (karyotype, mtDNA restriction fragment length polymorphism (RFLP) and microsatellite-primed PCR (MSP-PCR)) diversity of the conspecific strains indicated that diverse clones of the species coexisted in the wines. Genetic segregation of certain clones and interactions (antagonism and crossfeeding) of the species also appeared to shape the fermenting yeast biota.

Determination of Ochratoxin A and Ochratoxin B in Archived Tokaj Wines (Vintage 1959-2017) Using On-Line Solid Phase Extraction Coupled to Liquid Chromatography.

According to the EU legislation, ochratoxin A contamination is controlled in wines. Tokaj wine is a special type of sweet wine produced from botrytized grapes infected by "noble rot" Botrytis cinerea. Although a high contamination was reported in sweet wines and noble rot grapes could be susceptible to coinfection with other fungi, including ochratoxigenic species, no screening of Tokaj wines for mycotoxin contamination has been carried out so far. Therefore, we developed an analytical method for the determination of ochratoxin A (OTA) and ochratoxin B (OTB) involving online SPE coupled to HPLC-FD using column switching to achieve the fast and sensitive control of mycotoxin contamination. The method was validated with recoveries ranging from 91.6% to 99.1% with an RSD less than 2%. The limits of quantification were 0.1 and 0.2 µg L-1 for OTA and OTB, respectively. The total analysis time of the online SPE-HPLC-FD method was a mere 6 min. This high throughput enables routine analysis. Finally, we carried out an extensive investigation of the ochratoxin contamination in 59 Slovak Tokaj wines of 1959-2017 vintage. Only a few positives were detected. The OTA content in most of the checked wines did not exceed the EU maximum tolerable limit of 2 µg L-1, indicating a good quality of winegrowing and storing.

Physico-Chemical Characteristics and Culturable Microbial Communities of Grape Berries Change Strongly during Noble Rot Development.

Botrytis cinerea is a well-known pathogen of grapevine. However, under certain microclimatic conditions, Botrytis infection results in noble rot, an essential process in the production of the world-known Tokaji aszú wines in Hungary. We investigated the physico-chemical characteristics and culturable microorganisms associated with grape berries through several noble rot phases in the two main cultivars grown in Tokaj: Vitisvinifera cv. "Furmint" and "Hárslevelu". We measured physical and analytical parameters routinely tested in viticulture and analyzed the ITS rDNA sequence data of fungi isolated from the sampled berries. We observed significant differences in the physico-chemical parameters among the noble rot phases as well as sampling dates. The greatest variation in berry texture and microbial structure was observed in the initial phases, with variables converging as the noble rot progressed. By finding a bijection between the examined chemical properties and the factorial parameters (e.g., noble rot phase, collection time, cultivar), an appropriate sweet winemaking material can be designed. Fungal community differed significantly among cultivars, with higher number of species observed in Hárslevelu. Our results reveal that there is more to noble rot than only Botrytiscinerea and other microorganisms may play important roles in the aszú process.

Analysis of Low Cancer Mortality Rates in the Wine Regions of Tokaj and Balaton in Hungary.

The age-adjusted death rates (AADRs) due to cancers were investigated in two historical regions of white wines (Tokaj and Balaton) and in Hódmezovásárhely (HMV) as a control territory in Hungary between 2000 and 2010 evaluating 111,910 persons. The results of AADRs due to the eight most frequent types/gastrointestinal cancers were as follows: Tokaj 2120/664, Balaton: 2417/824, HMV: 2770/821, nationwide: 2773/887. The values found in Tokaj and Balaton regions were significantly less than those of HMV and nationwide. However, the least values were found in Tokaj. This Tokaj-related strong difference was not found among the regions in the case of young populations with hematological diseases but only in the older people who have been consuming their wines for decades. Supposedly, this wine-specific anti-cancer phenomenon could be related to the chemical differences existing in the two types of white wines, namely, to the pro-oxidant molecules of Tokaj wines derived from Botrytis cinerea. The roles of red meat consumption, hardness of drinking water, mineral content of soil, and socioeconomic status were negligible. It should be stressed that these data are valid only for these populations, for this period. Noteworthily, the different types of wines may have different effects on mortality rates during long-lasting consumptions.

Comparison of Different Categories of Slovak Tokaj Wines in Terms of Profiles of Volatile Organic Compounds.

The present work deals with the characterization of volatile organic compounds (VOCs) in wines from the Slovak Tokaj wine region. Studied wine samples were divided into three groups-varietal wines from registered Tokaj vine varieties, film wines Tokajské samorodné dry, and naturally sweet botrytized wines Tokaj selections. The VOCs from wines were extracted using optimized solid phase microextraction (SPME) and analyzed by comprehensive two-dimensional gas chromatography (GC×GC) coupled to high-resolution time-of-flight mass spectrometry (HRTOF-MS). In total, 176 VOCs were identified in all 46 studied samples. It was found that the total number of VOCs in varietal wines was generally higher than in botrytized wines. All three studied categories showed characteristic VOC profiles with significant differences. Varietal wines were characterized by higher concentrations of esters and terpenoids originating from grapes. The presence of γ-octalactone, (E)-6-methylhept-2-en-4-one, and lack of benzaldehyde were typical for Tokajské samorodné dry. Tokaj selections expressed the highest concentration of diethyl malate, benzaldehyde, and furfurals. Several interesting trends were also observed. The concentration of fermentation products was highest in varietal wines, while long-term matured Tokaj special wines were typified by the presence of compounds related to noble-rotten raisins (2-phenylacetaldehyde, ethyl 2-phenylacetate, and 2-phenylethanol), wood (cis-whisky lactone), and aging (1,1,6-trimethyl-2H-naphthalene, furfural, and 5-methylfurfural).

[The standardized mortality numbers of patients with gastrointestinal tumors and cardiovascular diseases in four wine regions and in one not-wine region of Hungary between 2000-2010]. / A gastrointestinalis daganatok és szív-ér rendszeri betegségek standardizált halálozási arányszámai Magyarország négy borvidékén és egy nem borvidéken 2000­2010 között.

INTRODUCTION: Eating habits act on mortalities from gastrointestinal tumors and cardiovascular diseases. AIM: To investigate the role of wine drinking on these mortalities in Hungary. METHOD: The standardized mortality data of people from 206,159 subjects died of gastrointestinal tumors and cardiovascular diseases between 2000-2010 were compared in four wine regions: Tokaj (white), Eger (red), Balaton (white), Szekszárd/Villány (red) and in Hódmezovásárhely (not-wine region). RESULTS: The significantly smallest number of tumors (664) occurred in Tokaj, but the cardiovascular mortality here was the highest (5955). On the other hand, the fewest cardiovascular mortality occurred in Szekszárd/Villány (3907), but showing here (831) and in Eger (934) the highest values of tumor death. CONCLUSIONS: The protective effect of red wine on cardiovascular mortality was verified. Surprisingly, the low value of gastrointestinal mortality in "Tokaj" - besides the higher level of selenium in tap water - shows some hidden features of these white wines. Orv Hetil. 2017; 158(25): 992-998.

Overwintering of Vineyard Yeasts: Survival of Interacting Yeast Communities in Grapes Mummified on Vines.

The conversion of grape must into wine involves the development and succession of yeast populations differing in species composition. The initial population is formed by vineyard strains which are washed into the must from the crushed grapes and then completed with yeasts coming from the cellar environment. As the origin and natural habitat of the vineyard yeasts are not fully understood, this study addresses the possibility, that grape yeasts can be preserved in berries left behind on vines at harvest until the spring of the next year. These berries become mummified during the winter on the vines. To investigate whether yeasts can survive in these overwintering grapes, mummified berries were collected in 16 localities in the Tokaj wine region (Hungary-Slovakia) in early March. The collected berries were rehydrated to recover viable yeasts by plating samples onto agar plates. For the detection of minority species which would not be detected by direct plating, an enrichment step repressing the propagation of alcohol-sensitive yeasts was also included in the process. The morphological, physiological, and molecular analysis identified 13 basidiomycetous and 23 ascomycetous species including fermentative yeasts of wine-making relevance among the 3879 isolates. The presence of viable strains of these species demonstrates that the grapes mummified on the vine can serve as a safe reservoir of yeasts, and may contribute to the maintenance of grape-colonizing yeast populations in the vineyard over years, parallel with other vectors and habitats. All basidiomycetous species were known phylloplane yeasts. Three Hanseniaspora species and pigmented Metschnikowia strains were the most frequent ascomycetes. Other fermentative yeasts of wine-making relevance were detected only in the enrichment cultures. Saccharomyces (S. paradoxus, S. cerevisiae, and S. uvarum) were recovered from 13% of the samples. No Candida zemplinina was found. The isolates with Aureobasidium morphology turned out to belong to Aureobasidium subglaciale, Kabatiella microsticta, or Columnosphaeria fagi. The ascomyceteous isolates grew at high concentrations of sugars with Wickerhamomyces anomalus being the most tolerant species. Complex interactions including antagonism (growth inhibition, contact inhibition, competition for nutrients) and synergism (crossfeeding) among the isolates and with Botrytis cinerea shape the composition of the overwintering communities.