The Riboflavin Metabolism in Four Saccharomyces cerevisiae Wine Strains: Assessment in Oenological Condition and Potential Implications with the Light-Struck Taste.

Riboflavin (RF), or vitamin B2, is an essential compound for yeast growth and a precursor of the flavin coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in redox and non-redox processes. RF is a photosensitive compound involved in the light-struck taste (LST), a fault causing the formation of off-flavors that can develop when the wine is exposed to light in the presence of methionine (Met), as well. As both RF and Met can be associated with detrimental changes in wines, a better comprehension of its yeast-mediated production is relevant to predict the maintenance of the desired character of the wine. This study aims at assessing the production of flavin derivatives (FDs) and Met by S. cerevisiae oenological starters under laboratory conditions. The results showed the presence of extra- and intracellular FDs, and Met is a strain-dependent characteristic being also affected by the initial content of RF in the medium. This finding was confirmed when the winemaking was carried out in a relevant environment. Our results evidenced the important impact of the yeast strain on the content of RF and its derivatives.

Evaluation of oenological tannins for preventing the light-struck taste.

The light-struck taste (LST) is a fault occurring in white and rosé wines associated to the formation of volatile sulfur compounds (VSCs) due to the reactions between riboflavin (RF) and methionine (Met). We investigated the possible preventing effect of 15 commercial tannins of different origin in model wine added with RF and Met, under oxic and anoxic conditions, and submitted to standardized light-exposure. All the tannins limited the degradation of Met in comparison to the tannin-free samples. Lower concentrations of VSCs were found in presence of tannins even under anoxia, condition favouring their formation. The sniffing trials evidenced the minor perception of cooked cabbage note with added tannins. The multivariate analysis showed the presence of flavan-3-ols was related to the formation of DMDS and DMTS, while tannic acid was related to MeSH, both the behaviors occurring under oxic condition. The study highlighted the ability of tannins to prevent the LST.

Response Surface Methodology Approach to Evaluate the Effect of Transition Metals and Oxygen on Photo-Degradation of Methionine in a Model Wine System Containing Riboflavin.

A Box-Behnken experimental design was implemented in model wine (MW) to clarify the impact of copper, iron, and oxygen in the photo-degradation of riboflavin (RF) and methionine (Met) by means of response surface methodology (RSM). Analogous experiments were undertaken in MW containing caffeic acid or catechin. The results evidenced the impact of copper, iron, and oxygen in the photo-induced reaction between RF and Met. In particular, considering a number of volatile sulfur compounds (VSCs) that act as markers of light-struck taste (LST), both transition metals can favor VSC formation, which was shown for the first time for iron. Oxygen in combination can also affect the concentration of VSCs, and a lower content of VSCs was revealed in the presence of phenols, especially caffeic acid. The perception of "cabbage" sensory character indicative of LST can be related to the transition metals as well as to the different phenols, with potentially strong prevention by phenolic acids.

Light-Struck Taste in White Wine: Protective Role of Glutathione, Sulfur Dioxide and Hydrolysable Tannins.

Light exposure of white wine can cause a light-struck taste (LST), a fault induced by riboflavin (RF) and methionine (Met) leading to the formation of volatile sulfur compounds (VSCs), including methanethiol (MeSH) and dimethyl disulfide (DMDS). The study aimed to investigate the impact of different antioxidants, i.e., sulfur dioxide (SO2), glutathione (GSH) and chestnut tannins (CT), on preventing LST in model wine (MW) and white wine (WW), both containing RF and Met. Both MW and WW samples were added with the antioxidants, either individually or in different combinations, prior to 2-h light exposure and they were stored in the dark for 24 months. As expected, the light induced the degradation of RF in all the conditions assayed. Met also decreased depending on the antioxidants added. The presence of antioxidants limited the formation of LST as lower concentrations of VSCs were found in both MW and WW samples. In the latter matrix, neither MeSH nor DMDS were detected in the presence of CT, while only DMDS was found in WW+GSH, WW+SO2+GSH and WW+CT+SO2 samples at a concentration lower than the perception thresholds. Considering the antioxidants individually, the order of their effectiveness was CT ≥ GSH > SO2 in WW under the adopted experimental conditions. The results indicate tannins as an effective enological tool for preventing LST in white wine and their use will be further investigated in different white wines under industrial scale.

From plant resistance response to the discovery of antimicrobial compounds: The role of volatile organic compounds (VOCs) in grapevine downy mildew infection.

The discovery of new mechanisms of resistance and natural bioactive molecules could be two of the possible ways to reduce fungicide use in vineyard and assure an acceptable and sustainable protection against Plasmopara viticola, the grapevine downy mildew agent. Emission of volatile organic compounds (VOCs), such as terpenes, norisoprenoids, alcohols and aldehydes, is frequently induced in plants in response to attack by pathogens, such as P. viticola, that is known to cause a VOCs increment in cultivars harboring American resistance traits. In this study, the role of leaf VOCs in the resistance mechanism of two resistant cultivars (Mgaloblishvili, a pure Vitis vinifera cultivar, and Bianca, an interspecific hybrid) and the direct antimicrobial activity of four selected VOCs have been investigated. The leaf VOCs profiles, analyzed through solid-phase microextraction gas chromatography-mass spectrometry analysis, as well as the expression of six terpene synthases (TPSs), were determined upon pathogen inoculation. In both cultivars, the expression pattern of six TPSs increased soon after pathogen inoculation and an increment of nine VOCs has been detected. While in Mgaloblishvili VOCs were synthesized early after P. viticola inoculation, they constituted a late response to pathogen in Bianca. All the four terpenes (farnesene, nerolidol, ocimene and valencene), chosen according to the VOC profiles and gene expression analysis, caused a significant reduction (53-100%) in P. viticola sporulation. These results support the role of VOCs into defense mechanisms of both cultivars and suggest their potential role as a natural and eco-friendly solution to protect grapevine from P. viticola.

Oxygen-induced faults in bottled white wine: A review of technological and chemical characteristics.

Several changes can take place in wine after blotting. Some of them lead to the desired evolution of wine being more complex, round and pleasant. However, unexpected changes can also occur ascribable to the premature wine oxidation (PremOx) arising when a wine, presumably with aging potential, results oxidized and often undrinkable. The complexity of PremOx, where aromas are also involved, makes difficult to identify all the oxidation products, and to predict its occurrence in wines. Despite most studies have been focused on the effect of time after wine bottling on PremOx as well as pinking phenomena, identification of pinking markers, reliable methods for their detection in wine, and correlations between markers and the wine-bottle-closure system are still unknown. This review aimed to highlight aspects PremOx-related, including wine-bottle-closure system, color change, with particular emphasis on pinking, and aroma decay based on the current knowledge becoming the bases for future perspectives.

Polyphenol Bioavailability and Plasma Antiradical Capacity in Healthy Subjects after Acute Intake of Pigmented Rice: A Crossover Randomized Controlled Clinical Trial.

Health-promoting effects of plant foods have been emphasized in the last few decades and ascribed to the bioactive phytochemicals present therein-in particular, phenylpropanoids. The latter have been investigated for a number of preclinical biological activities, including their antioxidant power. Due to the paucity of human studies, in this randomized intervention trial, we investigated whether the acute intake of pigmented rice could increase the plasma bioactive levels and antiradical power in twenty healthy subjects. A crossover randomized controlled clinical trial was conducted on 19 volunteers. Artemide and Venere black rice cultivars were tested, while brown rice Carnaroli was used as a control. Each patient received randomly one serving (100 g) of rice on three different experimental days, separated by a 7-day washout period. After baseline blood withdrawal, time-course changes of plasma polyphenols, flavonoids and radical-scavenging capacity were determined at 30, 60, 120 and 180 min post rice intake. Compared to Carnaroli rice, the two black rice cultivars significantly increased the plasma levels of polyphenols and flavonoids at 60 and 120 min and, correspondingly, the plasma antiradical power at 60 min after consumption. Pigmented rice consumption can contribute to diet-related health benefits in humans.

Investigating the Role of Antioxidant Compounds in Riboflavin-Mediated Photo-Oxidation of Methionine: A 1H-NMR Approach.

Riboflavin (RF) is a well-known photosensitizer, responsible for the light-induced oxidation of methionine (Met) leading to the spoilage of wine. An NMR approach was used to investigate the role of gallic acid (GA) and sulfur dioxide (SO2) in the RF-mediated photo-oxidation of Met. Water solutions of RF and Met, with and without GA or SO2, were exposed to visible light for increasing time in both air and nitrogen atmospheres. Upon light exposure, a new signal appeared at 2.64 ppm that was assigned to the S(O)CH3 moiety of methionine sulfoxide. Its formation rate was lower in a nitrogen atmosphere and even lower in the presence of GA, supporting the ability of this compound in quenching the singlet oxygen. In contrast, SO2 caused relevant oxidation of Met, moderately observed even in the dark, making Met less available in donating electrons to RF. The competition of GA versus Met photo-oxidation was revealed, indicating effectiveness of this antioxidant against the light-dependent spoilage of wine. A pro-oxidant effect of SO2 toward Met was found as a possible consequence of radical pathways involving oxygen.

Impact of Cooking on Bioactive Compounds and Antioxidant Activity of Pigmented Rice Cultivars.

Pigmented rice cultivars, namely Venere and Artemide, are a source of bioactive molecules, in particular phenolics, including anthocyanins, exerting a positive effect on cardiovascular systems thanks also to their antioxidant capacity. This study aimed to determine the total phenol index (TPI), total flavonoids (TF), total anthocyanins (TA) and in vitro antioxidant capacity in 12 batches of Venere cultivar and two batches of Artemide cultivar. The rice was cooked using different methods (boiling, microwave, pressure cooker, water bath, rice cooker) with the purpose to individuate the procedure limiting the loss of bioactive compounds. TPI, TF and TA were spectrophotometrically determined in both raw and cooked rice samples. Rice samples of Artemide cultivars were richer in TPI (17.7-18.8 vs. 8.2-11.9 g gallic acid/kg in Venere rice), TF (13.1 vs. 5.0-7.1 g catechin/kg rice for Venere rice) and TA (3.2-3.4 vs. 1.8-2.9 g Cy-3glc/kg for Venere rice) in comparison to those of Venere cultivar; as well, they showed higher antioxidant capacity (46.6-47.8 vs. 14.4-31.9 mM Trolox/kg for Venere rice). Among the investigated cooking methods, the rice cooker and the water bath led to lower and comparable losses of phenolics. Interestingly, the cooking water remaining after cooking with the rice cooker was rich in phenolics. The consumption of a portion of rice (100 g) cooked with the rice cooker with its own cooking water can supply 240 mg catechin and 711 mg cyanidin 3-O-glucoside for Venere rice and 545 mg catechin and 614 mg cyanidin 3-O-glucoside for Artemide rice, with a potential positive effect on health.

Oxidations in white grape (Vitis vinifera L.) skins: Comparison between ripening process and photooxidative sunburn symptoms.

Oxidations in grape berries are gaining major interest as they affect grape characteristics and quality. Considering berries, Reactive Oxygen Species are involved in the responses to both ripening process and stresses, including photooxidative sunburn. Redox metabolism involves a multitude of chemical and enzymatic reactions. In this study, four white grape cultivars were examined for natural ripening and photooxidative sunburn effects (obtained in artificial conditions) on berry pigmentation, chemical composition and enzymatic activity. The measured parameters included reflectance spectra, pigmentation (including berry browning), content of photosynthetic pigments, organic acid profiles, antioxidant activity, concentrations of antioxidants (total phenolics, ascorbic acid and reduced glutathione), enzymatic activities (guaiacol peroxidases, ascorbate peroxidase and catalase). The effects of the treatment (natural ripening and artificial photooxidative sunburn) on each considered parameter are described in the paper. Photooxidative sunburn strongly affected the contents of antioxidants and chlorophylls, increased the browning index and modulated the enzymatic activities investigated. Samples clearly clustered depending on the oxidation status. Furthermore, the PCA highlighted the similarities and differences in the responses to oxidative stress during ripening and photooxidative sunburn. PCA produced five functions with eigenvalues higher than 1, representing 87.03% of the total variability. In particular, the scores of the function 1 discriminated the samples based on the oxidation status, while the function 2 separated the samples based on the sampling date, representing the physiological responses characteristic of ripening. Our work sheds light on this topic, and will allow a more conscious vineyard management, thus supporting the agricultural adaptation to climate changes.

Production of melatonin and other tryptophan derivatives by Oenococcus oeni under winery and laboratory scale.

Malolactic fermentation (MLF) in Valtellina Superiore DOCG red wine was monitored in 4 cellars and the final products were analysed to determine the content of melatonin (MEL) and other tryptophan (TRP) derivatives, including tryptophan ethyl ester (TEE) and MEL isomers (MISs), and to isolate predominant O. oeni strains. MEL and TEE significantly increased in wines after MLF from two cellars out of four. Six strains were isolated during the MLF of red wines and under laboratory scale, in rich and synthetic wine cultural media, together with other four O. oeni strains able to trigger the MLF. Results showed that the presence of stressful growth factors, like ethanol and acid pH, has a pivotal role in triggering the release of TEE by oenococci. Indeed, all the strains became capable to produce also MEL and MISs, together with TEE. under harsh growth conditions, as in a synthetic wine medium. The production of these compounds was strain-dependent and a maximum amount of 0.0078 ±â€¯0.0023 ngT/mL (UMB472) and 619.85 ±â€¯196.16 ngT/mL (UMB436) of MEL and TEE was obtained, respectively. In particular, different MISs were detected under oenological and laboratory scale suggesting that other factors (i.e. technological and/or physico-chemical) could affect the synthesis of TRP derivatives.

Light-induced reactions of methionine and riboflavin in model wine: Effects of hydrolysable tannins and sulfur dioxide.

The riboflavin-mediated photo-degradation of methionine in white wine has been related to onset of undesired light-struck taste. This research investigated the effects of different concentrations of riboflavin and methionine, hydrolysable tannins from various sources (nut galls, chestnut and oak woods) and sulfur dioxide on methionine degradation in a model wine exposed to light. Increased methionine concentration resulted in its increased degradation with the consequent formation of volatile sulfur compounds, namely methanethiol, dimethyl disulphide and dimethyl trisulphide. Tannins, especially nut gall tannin, were effective in limiting both methionine degradation and the production of volatile sulfur compounds. Sulfur dioxide enhanced the methionine degradation although the light-struck taste was not perceived when sulfur dioxide concentration was higher than 50 mg/L. In conclusion, the use of hydrolysable tannins can represent a promising tool for protecting white wine against the light-struck taste also limiting the use of sulfur dioxide.

Assessment of Tryptophan, Tryptophan Ethylester, and Melatonin Derivatives in Red Wine by SPE-HPLC-FL and SPE-HPLC-MS Methods.

Melatonin (MEL) is an indoleamine produced mainly by the pineal gland in vertebrates. It plays a significant role in the regulation of circadian rhythms, mitigation of sleeping disorders, and jet lag. This compound is synthetized from tryptophan (TRP) and it has been found in seeds, fruits, and fermented beverages, including wine. Wine is also a source of other tryptophan derivatives, the tryptophan ethylester (TEE) and MEL isomers (MISs), for which the biological properties need to be elucidated. An analytical method for the simultaneous quantification of TRP, TEE, and MEL was developed by a Solid Phase Extraction (SPE) of a preconcentration of wine followed by high performance liquid chromatography (HPLC) analysis either with fluorescence or mass spectrometer detectors. The analytical method showed a relative standard deviation (RSD) lower than 8%, except for TRP (RSD 10.5% in wine). The recovery was higher than 76%. The versatility of SPE preconcentrations allowed for the adequate preconcentration of wine sample as well as detection of low concentrations, an important aspect especially for MEL (detection limit 0.0023 µg/L). The proposed method proved to be suitable for assessing the investigated compounds in some red wine samples, where 74.4⁻256.2 µg/L and 0.038⁻0.063 µg/L of TEE and MEL were detected, respectively. Five MISs were also found in wine samples in concentrations up to 1.97 µg/L.

Brettanomyces bruxellensis yeasts: impact on wine and winemaking.

Yeasts belonging to the Brettanomyces/Dekkera genus are non-conventional yeasts, which affect winemaking by causing wine spoilage all over the world. This mini-review focuses on recent results concerning the presence of Brettanomyces bruxellensis throughout the wine processing chain. Here, culture-dependent and independent methods to detect this yeast on grapes and at the very early stage of wine production are encompassed. Chemical, physical and biological tools, devised for the prevention and control of such a detrimental species during winemaking are also presented. Finally, the mini-review identifies future research areas relevant to the improvement of wine safety and sensory profiles.

Yeast contribution to melatonin, melatonin isomers and tryptophan ethyl ester during alcoholic fermentation of grape musts.

Melatonin (MEL) has been found in some medicinal and food plants, including grapevine, a commodity of particular interest for the production of wine, a beverage of economic relevance. It has also been suggested that MEL in wine may, at least in part, contribute to the health-promoting properties attributed to this beverage and, possibly, to other traditional Mediterranean foodstuffs. After a preliminary screening of 9 yeast strains in laboratory medium, three selected strains (Saccharomyces cerevisiae EC1118, Torulaspora delbrueckii CBS1146(T) and Zygosaccharomyces bailii ATCC36947(T) ) were inoculated in experimental musts obtained from 2 white (Moscato and Chardonnay) and 2 red (Croatina and Merlot) grape varieties. The production of MEL, melatonin isomers (MIs) and tryptophan ethyl ester (TEE) was monitored during the alcoholic fermentation. The screening showed that the three investigated strains produced the highest concentrations of MEL and two MIs in optimal growth conditions. However, MEL and MIs were not produced in oenological conditions, but the three strains synthesized high concentrations of a new MI and TEE in musts.

Over-evaluation of total flavonoids in grape skin extracts containing sulphur dioxide.

Sulphur dioxide (SO2) proved to increase absorbance at 280 nm of grape skin and seed extracts containing it, diluted with ethanol-HCl to assess total flavonoids and anthocyanins in the same analysis. Additional absorbance at 280 nm was also observed in acetone:H2O extracts, if the acetone had not completely evaporated before the extracts were diluted with a solvent. Flavonoids were correctly quantified in the extracts when SO2 or acetone were removed by solid-phase extraction with a C18 RP as sorbent and methanol as eluting solvent.

Determination of sotolon content in South African white wines by two novel HPLC-UV and UPLC-MS methods.

Sotolon has been reported to play an important role in the atypical ageing and aroma character of many wines. A number of analytical techniques for sotolon analysis in wine have been reported, but these often require extensive sample preparation. In this work we report a HPLC-UV method and a novel UPLC-MS method to determine sotolon concentrations in white wines with little sample preparation applied for the first time for the evaluation of sotolon levels in South African wines. The validation showed that the instrumental methods had good accuracy, repeatability and linearity, but the UPLC-MS method proved more sensitive. For both methods, quantification limits were lower than the sotolon odour threshold in wine (10µg/L), 0.86µg/L and 0.013µg/L, for HPLC-UV and UPLC-MS methods, respectively. Sotolon levels in 65 South African white wines were often found to be lower than the reported odour threshold, with the highest concentration being 9.11µg/L. However, for low levels (<1µg/L), unknown interferences in certain wines led to sotolon not being quantified with the HPLC-UV method, which made the UPLC-MS method more suitable.

UHPLC quantification of sotolon in white wine.

Sotolon (4,5-dimethyl-3-hydroxy-2,5-dihydrofuran-2-one) is a volatile compound involved in the atypical aging of dry white wine, causing an irreversible defect when it exceeds 7-8 µg L(-1), and it might be adopted as a chemical marker of oxidative aging. An easier and sensitive ultrahigh-pressure liquid chromatography method for its determination in white wine is reported. The sample preparation is based on the liquid/liquid extraction by dichloromethane and the purification by solid phase extraction of the redissolved dry sample. This method showed good linearity and intermediate repeatability (<0.7 µg L(-1)), high recovery (>89.5%), and low detection limit (0.029 µg L(-1)). This method was usefully applied to 30 Italian sparkling and still white wine samples, where sotolon was not detected in most of them and exceeded the perception threshold only in one sparkling wine (13 µg L(-1)). The proposed method could be used to further investigate the aging/storage conditions and the chemical-physical parameters affecting its formation in wine.

Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis.

The effect of different sulphur dioxide concentrations on culturability and viability of seven strains of Brettanomyces bruxellensis was tested in a synthetic wine medium (SWM) and a different response to molecular SO(2) among strains was detected. Sulphur dioxide induced a viable but non culturable (VBNC) state in all the strains. The greater percentage of VBNC cells were identified for five strains at molecular SO(2) concentrations of 0.2mg/L and for two strains at the concentration of 0.4mg/L. Vinyl phenols were detected in media containing VBNC or not viable B. bruxellensis, suggesting that its spoilage metabolism could be maintained during wine storage. Overall, this study indicates that SO(2) is a chemical stressor inducing VBNC state in B. bruxellensis grown in synthetic wine medium. Further studies are needed to evaluate the effects of SO(2) on the metabolism of this yeast in wine spoilage.

Determination of reduced cysteine in oenological cell wall fractions of Saccharomyces cerevisiae.

Compounds containing cysteine residues, such as glutathione, can affect the redox potential of must and wine by reduction of o-quinones and hydrogen peroxide. The oenological yeast cell wall fractions contain cysteine residues in their protein structure, and they could affect both oxidative and odor properties of wine. An analytical approach based on the derivatization of cysteinyl residues with p-benzoquinone followed by reversed-phase high-performance liquid chromatography separation was developed to quantify glutathione and free and protein cysteine in 16 Saccharomyces cerevisiae strains and 12 commercial samples of yeast mannoproteins, hulls, and lysates. The chemical modifications induced by the Maillard reaction following the industrial preparation of such fractions were evaluated as well. Lysates showed the highest protein cysteine content and high contents of glutathione and free cysteine. Mannoproteins showed an intense Maillard reaction (furosine >60 mg/100 g protein), and most of the samples were able to bind thiol compounds with a potentially detrimental effect toward the thiol-related odors in wine.