Deep reinforcement learning classification of sparkling wines based on ICP-MS and DOSY NMR spectra.

An approach that combines NMR spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) and advanced tensor decomposition algorithms with state-of-the-art deep learning procedures was applied for the classification of Croatian continental sparkling wines by their geographical origin. It has been demonstrated that complex high-dimensional NMR or ICP-MS data cannot be classified by higher-order tensor decomposition alone. Extension of the procedure by deep reinforcement learning resulted in an exquisite neural network predictive model for the classification of sparkling wines according to their geographical origin. A network trained on half of the sample set was able to classify even 94% of all samples. The model can particularly be useful in cases where the number of samples is limited and when simpler statistical methods fail to produce reliable data. The model can further be exploited for the identification and differentiation of sparkling wines including a high potential for authenticity or quality control.

Garlic Ecotypes Utilise Different Morphological, Physiological and Biochemical Mechanisms to Cope with Drought Stress.

Drought negatively affects plants by altering morphological, physiological and metabolic processes and ultimately reducing yields. Garlic (Allium sativum L.), an important member of the Alliaceae family, is also sensitive to drought and maximizing the yield of garlic bulbs is largely dependent on water availability. The objective of this study was to determine the effects of drought stress on morphological and physiological characteristics, as well as on phenolic, sugar, inulin and free amino acid content and antioxidant activity in two Croatian garlic ecotypes, 'Istarski crveni' (IC) and Istarski bijeli (IB). Drought was induced by using polyethylene glycol 8000 (PEG) solution (-0.6 MPa) starting 21 days after clove planting and lasted for 20 days. Drought reduced plant height, number of leaves and plant weight, but increased root length in both ecotypes compared to the control treatment. Among the physiological parameters, significant differences were observed between the two ecotypes studied in the spectral characteristics of the leaves, namely reflection in red, green and blue, VAL, values of the vegetation indices related to the chlorophyll content (CHI, GI), and the anthocyanin content (ARI). Ecotype IC showed higher antioxidant activity in the control treatment due to higher total phenolic content (TPC), but under drought conditions higher DPPH radical scavenging activity was determined in ecotype IB and higher values of FRAP in IC. Sucrose and glucose generally decreased under drought, while inulin increased in IB but decreased in IC. Total free amino acid content increased under drought in both ecotypes. In conclusion, drought tolerance of IB might be associated with increased accumulation of inulin and higher levels of amino acids, especially those shown to contribute to drought resistance. In IC, drought tolerance is associated with an increase in some amino acid compounds and better root growth in depth, probably due to a more efficient translocation of sucrose to the underground part of the plant.

Croatian Native Grapevine Varieties' VOCs Responses upon Plasmopara viticola Inoculation.

The Plasmopara viticola pathogen causes one of the most severe grapevine diseases, namely downy mildew. The response to P. viticola involves both visible symptoms and intricate metabolomic alterations, particularly in relation to volatile organic compounds, and depends on the degree of resistance of a particular variety. There are numerous native grapevine varieties in Croatia, and they vary in susceptibility to this oomycete. As previously reported, in vitro leaf disc bioassay and polyphenolic compound analysis are complementary methods that can be used to separate native varieties into various resistance classes. This research used the Solid Phase Microextraction-Arrow Gas Chromatography-Mass Spectrometry method to identify the early alterations in the VOCs in the leaves after P. viticola inoculation. Based on the absolute peak area of sesquiterpenes, some discrepancies between the sampling terms were noticed. The presence of certain chemical compounds such as humulene, ylangene, and α-farnesene helped distinguish the non-inoculated and inoculated samples. Although specific VOC responses to P. viticola infection of native varieties from various resistance classes could not be identified, the response of less susceptible native varieties and resistant controls was associated with an increase in the absolute peak area of several compounds, including geranylacetone, ß-ocimene, and (E)-2-hexen-1-ol.

Discrimination of genetic and geographical groups of grape varieties (Vitis vinifera L.) based on their volatile organic compounds.

Grape volatile organic compounds (VOCs) play an important role in the winemaking industry due to their contribution to wine sensory characteristics. Another important role in the winemaking industry have the grapevine varieties used in specific regions or countries for wine production. Due to the high variability of grapevine germplasm, grapevine varieties are as classified based on their genetic and geographical origin into genetic-geographic groups (GEN-GEO). The aim of this research was to investigate VOCs in 50 red grapevine varieties belonging to different GEN-GEO groups. The study included varieties from groups C2 (Italy and France), C7 (Croatia), and C8 (Spain and Portugal). The analysis of VOCs was performed by SPME-Arrow-GC/MS directly from grape skins. The analyzed VOCs included aldehydes, ketones, acids, alcohols, monoterpenes, and sesquiterpenes. The most abundant VOCs were aldehydes and alcohols, while the most numerous were sesquiterpenes. The most abundant compounds, aldehydes and alcohols, were found to be (E)-2-hexenal, hexenal, (E)-2-hexen-1-ol, and 1-hexanol. Using discriminant analysis, the GEN-GEO groups were separated based on their volatile profile. Some of the individual compounds contributing to the discrimination were found in relatively small amounts, such as benzoic acid, (E,E)-2,4-hexadienal, 4-pentenal, and nonanoic acid. The groups were also discriminated by their overall volatile profile: group C2 was characterized by a higher content of aldehydes and alcohols, and group C8 was characterized by a higher content of sesquiterpenes and acids. Group C7 was characterized by all low amount of all classes of VOCs.

Leaf Polyphenolic Profile as a Determinant of Croatian Native Grapevine Varieties' Susceptibility to Plasmopara viticola.

Since grapevine is highly susceptible to various pathogens, enormous amounts of pesticides are applied each season to achieve profitable production. One of the most destructive grapevine diseases is downy mildew, and their interaction has been in the spotlight for more than a decade. When it comes to a metabolome level, phenolic compounds are relevant to investigate due to their involvement in the plant immune system and known antifungal properties. Croatian grapevine germplasm is highly heterogeneous due to its long history of cultivation in diversified geographical regions. Since it has been found that native varieties react differently to the infection of Plasmopara viticola, the intention of this study is to define if the chemical background of the leaves, i.e., polyphenolic composition, is responsible for these dissimilarities. Therefore, the leaves of 17 genotypes, among which 14 were native and 3 were controls, were analyzed using high-performance liquid chromatography (HPLC) in four terms: before inoculation and 24, 48, and 96 h post inoculation (hpi). During this early phase, significant differences were found neither between the terms nor between the non-inoculated and inoculated samples, except for resveratrol-3-O-glucoside. By applying principal component analysis (PCA) using initial leaf polyphenolic composition, varieties of V. vinifera were clearly separated into three different groups corresponding to their International Organization of Vine and Wine (OIV) classes of susceptibility to P. viticola. Results obtained in this research suggest that the initial constitutive polyphenolic composition of the cultivar leaves has a crucial influence on their susceptibility to P. viticola, and this finding can be used to improve the success of grapevine breeding programs toward downy mildew resistance.

Optimization of SPME-Arrow-GC/MS Method for Determination of Free and Bound Volatile Organic Compounds from Grape Skins.

(1) Background: Solid phase microextraction (SPME)-Arrow is a new extraction technology recently employed in the analysis of volatiles in food materials. Grape volatile organic compounds (VOC) have a crucial role in the winemaking industry due to their sensory characteristics of wine.; (2) Methods: Box-Behnken experimental design and response surface methodology were used to optimise SPME-Arrow conditions (extraction temperature, incubation time, exposure time, desorption time). Analyzed VOCs were free VOCs directly from grape skins and bound VOCs released from grape skins by acid hydrolysis.; (3) Results: The most significant factors were extraction temperature and exposure time for both free and bound VOCs. For both factors, an increase in their values positively affected the extraction efficiency for almost all classes of VOCs. For free VOCs, the optimum extraction conditions are: extraction temperature 60 °C, incubation time 20 min, exposure time 49 min, and desorption time 7 min, while for the bound VOCs are: extraction temperature 60 °C, incubation time 20 min, exposure time 60 min, desorption time 7 min.; (4) Conclusions: Application of the optimized method provides a powerful tool in the analysis of major classes of volatile organic compounds from grape skins, which can be applied to a large number of samples.

Screening of Croatian Native Grapevine Varieties for Susceptibility to Plasmopara viticola Using Leaf Disc Bioassay, Chlorophyll Fluorescence, and Multispectral Imaging.

In the era of sustainable grapevine production, there is a growing demand to define differences between Vitis vinifera varieties in susceptibility to downy mildew. Croatia, as a country with a long tradition of grapevine cultivation, preserves a large number of native grapevine varieties. A leaf disc bioassay has been conducted on 25 of them to define their response to downy mildew, according to the International Organisation of Vine and Wine (OIV) descriptor 452-1, together with the stress response of the leaf discs using chlorophyll fluorescence and multispectral imaging with 11 parameters included. Time points of measurement were as follows: before treatment (T0), one day post-inoculation (dpi) (T1), two dpi (T2), three dpi (T3), four dpi (T4), six dpi (T5), and eight dpi (T6). Visible changes in form of developed Plasmopara viticola (P. viticola) sporulation were evaluated on the seventh day upon inoculation. Results show that methods applied here distinguish varieties of different responses to downy mildew. Based on the results obtained, a phenotyping model in the absence of the pathogen is proposed, which is required to confirm by conducting more extensive research.

Grapevine as a Rich Source of Polyphenolic Compounds.

Grapes are rich in primary and secondary metabolites. Among the secondary metabolites, polyphenolic compounds are the most abundant in grape berries. Besides their important impacts on grape and wine quality, this class of compounds has beneficial effects on human health. Due to their antioxidant activity, polyphenols and phenolic acids can act as anti-inflammatory and anticancerogenic agents, and can modulate the immune system. In grape berries, polyphenols and phenolic acids can be located in the pericarp and seeds, but distribution differs considerably among these tissues. Although some classes of polyphenols and phenolic acids are under strict genetic control, the final content is highly influenced by environmental factors, such as climate, soil, vineyard, and management. This review aims to present the main classes of polyphenolic compounds and phenolic acids in different berry tissues and grape varieties and special emphasis on their beneficial effect on human health.

Volatile Profile Characterization of Croatian Commercial Sparkling Wines.

Commercial sparkling wine production represents a relatively low but important part of the Croatian wine production, especially in the Zagreb county. This study presents the results of volatile aroma compounds profile and organic acid composition of commercial sparkling wine samples from three vine-growing regions in Zagreb county. In total, 174 volatile aroma compounds were identified, separated between their chemical classes (aldehydes, higher alcohols, volatile phenols, terpenes, C13-norisoprenoids, lactones, esters, fatty acids, sulfur compounds, other compounds, other alcohols). Higher alcohols such as phenylethyl and isoamyl alcohol as well as 2-methyl-1-butanol, and esters such as diethyl succinate, ethyl hydrogensuccinate, and ethyl lactate had the strongest impact on the volatile compounds profile of Zagreb county sparkling wine. The presence of diethyl glutarate and diethyl malonate, compounds whose concentrations are influenced by yeast autolysis or caused by chemical esterification during the ageing process, was also noted. The influence of every single volatile aroma compound was evaluated by discriminant analysis using forward stepwise model. The volatile profiles of traditional sparkling wines from Croatia were presented for the first time. It is hoped the results will contribute to better understanding the quality potential and to evaluate possible differences on the bases of detected aroma concentrations and multivariate analysis.

Effect of Different Reducing Agents on Aromatic Compounds, Antioxidant and Chromatic Properties of Sauvignon Blanc Wine.

Sulfur dioxide (SO2) is widely the most used enological additive with reductive, antiseptic and dissolving properties. According to increasing health concerns and the gradual decrease in total SO2 concentrations allowed in wine, alternative and supplementary agents for preservation are being investigated. For this reason, the current study was focused on the impact of different commercial reductive agents on white wine antioxidant activity and chemical composition. The effect of additives that combine sulfites, ascorbic acid and enological tannins were compared against standard 5% sulfurous acid (H2SO3) during the pre-fermentative treatments of Sauvignon Blanc must (Vitis vinifera L.). The basic parameters of quality, free amino-nitrogen and total polyphenoliccompounds in must were analyzed. Gas chromatography and spectrophotometric methods were used to investigate the overall volatile composition, antioxidant and chromatic parameters in wines. The obtained results undoubtedly pointed out the positive effect of sulfuric acid on the fermentation dynamics. Furthermore, application of combined reducing additives with potassium metabisulfite, L-ascorbic acid, gallotannins and ellagitannins, resulted in a higher antioxidant capacity and increased concentration of aromatic compounds and their odor activity values in Sauvignon Blanc wine.

Solid-liquid Extraction of Phenolics from Red Grape Skins.

For the characterization of grape cultivars, the profile and content of flavonoids are important because these compounds have an impact on grape and wine quality. A new extraction method for the recovery of flavonoids, e.g. anthocyanins, flavonols and flavan-3-ols from grape skins was developed. The optimization of solid-liquid extraction of flavonoids was conducted, with respect to the type of the organic solvent and its percentage in the extraction solvent as well as the extraction temperature and extraction time, using response surface methodology. Optimal conditions were obtained by using extraction solvent composed from acetonitrile:water:formic acid (20:79:1; v/v/v), at an extraction temperature of 50 °C, an extraction time of 1 h in a single-step extraction and with a solid-to-solvent ratio of 1:80 g mL(-1) (125 mg of grape skin powder and 10 mL of extraction solvent). The new optimal extraction method is inexpensive, simple, fast, accurate and selective for the recovery of simple flavonoids.

Multi-response optimisation of ultrasound-assisted extraction for recovery of flavonoids from red grape skins using response surface methodology.

INTRODUCTION: For the characterisation of grape cultivars, the profile and content of flavonoids are important because these compounds impact grape and wine quality. To determine the correct profile and content of flavonoids, the use of robust, sensitive and reliable methods is necessary. OBJECTIVE: The object of this research is to develop a new ultrasound-assisted extraction (UAE) method for the recovery of flavonoids from grape skins using response surface methodology. METHOD: Optimisation of UAE was performed using a complementary study combining a Box-Behnken experimental design with qualitative analysis by high-performance liquid chromatography. RESULTS: Optimal extraction conditions were obtained using the extraction solvent composed of acetonitrile:water:formic acid (26:73:1, v/v/v) at an extraction temperature of 50 °C, an extraction time of 15 min in a single-extraction step and with a solid-to-solvent ratio of 1:80 g/mL. The calculated relative standard deviations for the optimal extraction method were very low, measuring less than 5%. CONCLUSIONS: This study demonstrates that numerous factors have strong effects on the extraction efficiency, including the type of organic modifier and its percentage in the extraction solvent, the number of extraction steps, the solid-to-solvent ratio, the extraction time and temperature and, finally, the particular nature of analyte and their position within the grape skin cell.

Application of pectinases for recovery of grape seeds phenolics.

Grape marc seeds contain large amounts of different polyphenolic compounds so they can be used for the recovery of these classes of compounds. A new green extraction method for the recovery of phenols from the grape seeds was developed. To provide a high quality extract sourced from natural product by using enzymes as alternative solvents and spending less energy, it is impeccable to call this method "green". Furthermore, this method was optimized by finding out which conditions provide the best possible results and achieving the maximum recovery of polyphenols from grape seeds. The optimization of the enzyme-assisted extraction of phenols was conducted using the commercially available oenological enzyme preparations with respect to the enzyme dosage, temperature, extraction time, pH value and enzyme preparation by applying the response surface methodology. Optimal conditions were determined using the enzyme preparation Lallzyme EX-V, at the extraction temperature of 48 °C, extraction time of 2 h and 43 min, pH 3.5 and enzyme dosage of 20.00 mg g-1. The new optimized extraction method is less expensive, simple, fast, precise and selective for the recovery of simple phenols (monomeric and dimeric form) and since it is based on the environmentally friendly extraction solvent it may provide a valuable alternative to the conventional extraction methods. The obtained extracts can be used for the application in pharmaceutical, food and cosmetic industry.