Characteristic phenolic composition of the Greek variety Mavrokountoura grape and wine

Mavrokountoura, a clone of the Mandilaria grape cultivar, was studied for the first time. The morphological characteristics and composition of berries were determined, as well as total phenols and tannins in skins, flesh, and seeds. The skin anthocyanins were identified using HPLC-MS/MS and quantified using HPLC-DAD. Two main anthocyanins were identified as malvidin-3-O-glucoside and malvidin-3-O-(6″-p-coumaroyl)-glucoside. One minor anthocyanin was identified as malvidin-3-O-(6″-acetyl)-glucoside. Glycosides of delphinidin, petunidin, and peonidin were detected in traces. Young Mavrokountoura wine was also studied. Extraction of total phenols from the grape to the young wine amounted to 32%, composing mainly non-tannic phenols. The individual anthocyanin components exhibited different extractability values from the skin to wine, ranging between 10–94%.     

Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production

The spoilage yeast Brettanomyces/Dekkera can persist throughout the winemaking process and has the potential to produce off-flavours that affect the sensory quality of wine. The main objective of this study was to select different strains of Brettanomyces bruxellensis isolated from red wines and to compare their volatile phenol production. From a collection of 63 strains, eight strains of B. bruxellensis were selected for volatile phenol production after the application of molecular techniques such as ISS–PCR, PCR–DGGE and REA–PFGE. All strains showed three large chromosomes of similar size with PFGE. However, unique restriction profiles of the chromosomes were visible after NotI digestion that clearly distinguished the strains. All strains were capable of producing large quantities of 4-ethylphenol and 4-ethylguaiacol from p-coumaric acid and ferulic acid, respectively in synthetic media. However, the diversity among strains for volatile phenol production differed between synthetic media and wine with regard to the maximum production levels of 4-ethylphenol and 4-ethylguaiacol. This study illustrated the diversity of B. bruxellensis strains that occur during winemaking.     

Identification and characterisation of anthocyanins in the antioxidant activity-containing fraction of Liriope platyphylla fruits

The objective of this study was to investigate antioxidant activities and anthocyanin profiles in the fruits of Liriope platyphylla, where these are considered functional substances in Korea. The acidic methanol extract of this species exhibited potent antioxidant activities, showing 83.9% DPPH scavenging activity and 92.5% ABTS scavenging activity at a concentration of 0.5 mg/ml. Moreover, anthocyanins were identified by reversed-phase C₁₈ column chromatography, NMR spectroscopy, and HPLC-DAD-ESI/MS analysis. Seven anthocyanins were characterised, including delphinidin-3-O-glucoside (1), delphinidin-3-O-rutinoside (2), cyanidin-3-O-glucoside (3), petunidin-3-O-glucoside (4), petunidin-3-O-rutinoside (5), malvidin-3-O-glucoside (6), and malvidin-3-O-rutinoside (7). Among these, petunidin-3-O-rutinoside (5) (7302.2 μg/g) and malvidin-3-O-rutinoside (7) (5776.1 μg/g) were the predominant anthocyanins, whereas the least prevalent anthocyanin was found to be cyanidin-3-O-glucoside (3) (64.9 μg/g). Therefore, our results suggest that strong antioxidant activities of the acidic methanol extract of L. platyphylla fruits are correlated with high anthocyanin contents, particularly the petunidin-3-O-rutinoside (5) and malvidin-3-O-rutinoside (7).     

Sorption of wine volatile phenols by yeast lees

The capacity of Saccharomyces cerevisiae yeast lees to sorb 4-ethylguaiacol and 4-ethylphenol was investigated in a synthetic medium and in wine. Active dried yeast was more effective when volatile phenols were diluted in red wine. Partition coefficients between wine model solution and wine yeast lees were determined and compared with those measured for dried active yeast. They showed a larger affinity of volatile phenols for wine yeast lees than for dried active yeast. The effect of yeast lees on volatile phenol sorption was sensitive to yeast autolysis level and to physicochemical parameters, such as ethanol content, temperature and pH. These results could be applied in the technology of reduction of organoleptic defects in wine due to phenols.     

Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma

Spoilage of red wine by the yeast species Dekkera bruxellensis is a common problem for the global wine industry. When conditions are conducive for growth of these yeasts in wine, they efficiently convert non-volatile hydroxycinnamic acids into aroma-active ethylphenols, thereby reducing the quality of the wine. It has been demonstrated previously that dissolved oxygen is a key factor which stimulates D. bruxellensis growth in wine. We demonstrate that whereas the presence of oxygen accelerates the growth of this species, oxygen-limited conditions favour 4-ethylphenol production. Consequently, we evaluated wine spoilage potential of three D. bruxellensis strains (AWRI1499, AWRI1608 and AWRI1613) under oxygen-limited conditions. Each strain was cultured in a chemically-defined wine medium and the fermentation products were analysed using HPLC and HS-SPME–GC/MS. The strains displayed different growth characteristics but were equally capable of producing ethylphenols. On the other hand, significant differences were observed for 18 of the remaining 33 metabolites analysed and duo-trio sensory analysis indicated significant aroma differences between wines inoculated with AWRI1499 and AWRI1613. When these wines were spiked with low concentrations of 4-ethylphenol and 4-ethylguaiacol, no sensorial differences could be perceived. Together these data suggest that the three predominant D. bruxellensis strains previously isolated during a large survey of Australian wineries do not differ substantively in their capacity to grow in, and spoil, a model wine medium.     

Anthocyanin composition in aged Chilean Cabernet Sauvignon red wines

Anthocyanins in aged Cabernet Sauvignon red wines were studied by HPLC–MS. The major anthocyanin in 6, 7, and 8 year old red wine extracts was the dimer vitisin A produced by condensation of malvidin-3-O-glucoside mediated by pyruvic acid. In aged wine, the content of malvidin-3-O-glucoside decreased with age with a concomitant increase of vitisin A. The latter is accompanied by several condensation products with molecular weight up to 1500 m/z. Differential pulse voltammetry indicated that aged wines have lower antioxidant capacity than young wine (400 mV), but higher than malvidin-3-O-glucoside (483 mV). Tafel’s plots showed that the electrochemical process occurring in aged wines is different from young wines. Six, seven and eight year old wines show similar behaviour with plots of 234, 177 and 188 mV/dec, respectively. These values are higher than the expected 120 mV/dec corresponding to a first electronic transfer but smaller compared to the 523 mV/dec corresponding to young wine.     

Chemical and antioxidative assessment of dietary turnip (Brassica rapa var. rapa L.)

The phenolic compounds and organic acids of turnip (Brassica rapa var. rapa L.) edible parts (leaves and stems, flower buds and roots) were determined by HPLC–DAD and HPLC–UV, respectively. The results revealed a profile composed of 14 phenolics (3-p-coumaroylquinic, caffeic, ferulic and sinapic acids, kaempferol 3-O-sophoroside-7-O-glucoside, kaempferol 3-O-sophoroside-7-O-sophoroside, kaempferol 3-O-(feruloyl/caffeoyl)-sophoroside-7-O-glucoside, kaempferol 3,7-O-diglucoside, isorhamnetin 3,7-O-diglucoside, kaempferol 3-O-sophoroside, 1,2-disinapoylgentiobiose, 1,2′-disinapoyl-2-feruloylgentiobiose, kaempferol 3-O-glucoside and isorhamnetin 3-O-glucoside) and six organic acids (aconitic, citric, ketoglutaric, malic, shikimic and fumaric acids). The quantification of the identified compounds showed kaempferol 3-O-sophoroside-7-O-glucoside, kaempferol 3-O-(feruloyl/caffeoyl)-sophoroside-7-O-glucoside, isorhamnetin 3,7-O-diglucoside and isorhamnetin 3-O-glucoside as the main phenolics, and malic acid as the organic acid present in highest amounts. A screening of the antioxidative potential was also performed by means of the DPPH radical scavenging assay. Turnip flower buds exhibited the strongest antioxidant capacity.     

Characterisation and preliminary bioactivity determination of Berberis boliviana Lechler fruit anthocyanins

Berberis boliviana Lechler is a member of the Berberidaceae family that has a small edible red-purple berry. The plant is native to the Peruvian Andes and contains high amounts of anthocyanin pigments. The monomeric anthocyanin content, determined by a pH-differential method, was 7/100 g of seedless berries. Pigments were characterised by HPLC coupled to a photodiode array (PDA) and mass spectrophotometer (MS) detectors. Five aglycones and ten anthocyanins were found and identified as petunidin-3-glucoside (24.4%), delphinidin-3-glucoside (24.1%), malvidin-3-glucoside (22.1%), cyanidin-3-glucoside (10.2%), petunidin-3-rutinoside (7.15%), malvidin-3-rutinoside (4.9%), cyanidin-3-rutinoside (3.8%), delphinidin-3-rutinoside (2.6%), peonidin-3-glucoside (1.1%), and peonidin-3-rutinoside (0.9%).     

Flavonoids, sugars and fruit acids of alpine bearberry (Arctostaphylos alpina) from Finnish Lapland

Alpine bearberry (Arctostaphylos alpina L.) is a special circumpolar edible berry primarily used for household purposes but with greater potential in commercial applications than utilized thus far. In this study, the flavonoids of alpine bearberry were investigated with HPLC-DAD and HPLC-MS, and the sugars and fruit acids as trimethylsilyl derivates with GC-FID. The most abundant anthocyanin was cyanidin-3-O-galactoside accounting for over 95% of the anthocyanins. Eight other anthocyanins existed in trace amounts only, the richest of them being cyanidin-3-O-arabinoside and cyanidin-3-O-glucoside. Cyanidin accounted for over 99% of the anthocyanin aglycones. All the flavonols identified were glycosides of quercetin. Majority of the sugars were glucose and fructose while the most abundant fruit acid was quinic acid. The unique anthocyanin composition of alpine bearberry encourages advanced use of the berry as a food ingredient in private households and in industrial applications. In addition, it serves as a unique reference material for research purposes.     

Pattern recognition of three Vitis vinifera L. red grapes varieties based on anthocyanin and flavonol profiles,with correlations between their biosynthesis pathways

The presence of anthocyanins and flavonols in three selected red grape varieties was investigated, in order to use their polyphenolic characterisation as a fingerprint. Berry skins of Gran Negro grapes were characterised by the presence of high content of malvidin- and peonidin-3-O-glucoside; Mouratón grapes, by the presence of high content of petunidin- and delphinidin-3-O-glucoside; and Brancellao grapes, by the presence of high content of cyanidin-3-O-glucoside. The main flavonols found included the 3-O-glucosides of quercetin, myricetin, kaempferol, laricitrin, isorhamnetin and syringetin. Using cluster analysis and principal components analysis, Gran Negro could be characterised by their content of isorhamnetin-3-O-glucoside and syringetin-3-O-glucoside and, along with Mouratón, by their myricetin conjugates. Flavonol profile could not provide a fingerprint of Brancellao variety. Stepwise discriminant analysis was performed in order to find the polyphenolic compounds, which characterised the selected grape varieties. Finally, anthocyanin and flavonol profiles in red grapes were compared and results confirmed that biosynthesis of flavonols is closely related to that of anthocyanins.     

Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts

Volatile phenols are aromatic compounds and one of the key molecules responsible for olfactory defects in wine. The yeast genus Brettanomyces is the only major microorganism that has the ability to covert hydroxycinnamic acids into important levels of these compounds, especially 4-ethylphenol and 4-ethylguaiacol, in red wine. When 4-ethylphenols reach concentrations greater than the sensory threshold, all wine’s organoleptic characteristics might be influenced or damaged. The aim of this literature review is to provide a better understanding of the physicochemical, biochemical, and metabolic factors that are related to the levels of p-coumaric acid and volatile phenols in wine. Then, this work summarizes the different methods used for controlling the presence of Brettanomyces in wine and the production of ethylphenols.     

Osmotic stress response in the wine yeast Dekkera bruxellensis

Dekkera bruxellensis is mainly associated with lambic beer fermentation and wine production and may contribute in a positive or negative manner to the flavor development. This yeast is able to produce phenolic compounds, such as 4-ethylguaiacol and 4-ethylphenol which could spoil the wine, depending on their concentration. In this work we have investigated how this yeast responds when exposed to conditions causing osmotic stress, as high sorbitol or salt concentrations. We observed that osmotic stress determined the production and accumulation of intracellular glycerol, and the expression of NADH-dependent glycerol-3-phosphate dehydrogenase (GPD) activity was elevated. The involvement of the HOG MAPK pathway in response to this stress condition was also investigated. We show that in D. bruxellensis Hog1 protein is activated by phosphorylation under hyperosmotic conditions, highlighting the conserved role of HOG MAP kinase signaling pathway in the osmotic stress response.     

The fate of flavanol–anthocyanin adducts in wines: Study of their putative reaction patterns in the presence of acetaldehyde

Methylmethine bridged flavanol–anthocyanin dimeric adducts (F–A) were synthesised by hemisynthesis using catechin, acetaldehyde and F–A adducts as precursors in order to simulate putative reactions undergoing in red wine. The products obtained, catechin–methylmethine–catechin–malvidin-3-O-glucose and methylmethine-(catechin–malvidin-3-O-glucose)₂, were analysed by HPLC–ESI-MS. The fragmentation patterns allowed the confirmation of the structures. These compounds were investigated and found in wine samples (2-year-old table wine and 10-year-old Port wine), thus confirming their formation in vino as a result of flavanol–anthocyanin adducts transformations.     

Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis

Brettanomyces bruxellensis is one of the most damaging species for wine quality, and tools for controlling its growth are limited. In this study, thirty-nine strains belonging to Saccharomyces cerevisiae and B. bruxellensis have been isolated from wineries, identified and then tested against a panel of thirty-nine killer yeasts. Here, for the first time, the killer activity of Ustilago maydis is proven to be effective against B. bruxellensis. Mixed cultures in winemaking conditions show that U. maydis CYC 1410 has the ability to inhibit B. bruxellensis, while S. cerevisiae is fully resistant to its killer activity, indicating that it could be used in wine fermentation to avoid the development of B. bruxellensis without undesirable effects on the fermentative yeast. The characterization of the dsRNAs isolated and purified from U. maydis CYC 1410 indicated that this strain produces a KP6-related toxin. Killer toxin extracts were active against B. bruxellensis at pH values between 3.0 and 4.5 and temperatures comprised between 15 °C and 25 °C, confirming their biocontrol activity in winemaking and wine aging conditions. Furthermore, small amounts (100 AU/ml) of killer toxin extracts from U. maydis significantly reduced the amount of 4-ethylphenol produced by B. bruxellensis, indicating that in addition to the growth inhibition observed for high killer toxin concentrations (ranging from 400 to 2000 AU/ml), small amounts of the toxin are able to reduce the production of volatile phenols responsible for the aroma defects in wines caused by B. bruxellensis.     

Mechanisms of anthocyanin degradation in grape must-like model solutions

Oxidative degradation of o-diphenolic (eg cyanidin-3-glucoside) and non-o-diphenolic (eg malvidin-3-glucoside) anthocyanins in the presence of caffeoyltartaric acid and grape polyphenoloxidase was studied in model solutions. Both anthocyanins reacted with the enzymically generated caffeoyltartaric acid o-quinone. Kinetic studies indicated that cyanidin-3-glucoside was degraded mostly by coupled oxidation whereas malvidin-3-glucoside formed adducts with caffeoyltartaric acid quinone. In solutions containing equimolar amounts of the anthocyanins, both reactions took place but coupled oxidation of cyanidin-3-glucoside occurred at a faster rate, partly protecting malvidin-3-glucoside. The occurrence of coupled oxidation resulted in sparing of caffeoyltartaric acid. Several oxidation products, including red pigments, were detected by HPLC. The UV-Vis spectra of the coloured condensation products suggested that they contained both caffeoyltartaric acid and anthocyanin moieties. All these pigments were gradually degraded to colourless compounds either by enzymatic oxidation or by further reaction with quinones.     

Copigmentation between malvidin-3-glucoside and some wine constituents and its importance to colour expression in red wine

Thermodynamic parameters for intermolecular copigmentation interactions involving malvidin-3-glucoside were determined by UV/visible spectroscopy at wine pH (pH 3.6). These included association constants, enthalpy and entropy changes, which were measured for chlorogenic acid, caffeic acid, quercetin, quercetin-3-glucoside, (−)-epicatechin, (+)-catechin, procyanidin dimer and seed tannin. Quercetin produced the strongest copigment (K_CP = 2900 ± 1300), whilst the addition of glucose at position 3 (quercetin-3-glucoside) reduced its effect by almost 10-fold. Malvidin-3-glucoside self-association (K_D = 3300 ± 300 mol⁻¹ l) was thermodynamically favoured over intermolecular interaction with any of the copigments tested. No colour enhancement due to self-association was observed for malvidin-3-glucoside-derived pigments that cannot enter hydration reactions. In addition, malvidin-3-(6-O-p-coumaryl)glucoside did not show colour enhancement suggesting that the p-coumaryl group prevents self-association. The malvidin-3-glucoside circular dichroism (CD) spectrum was not affected by indicated changes in malvidin-3-glucoside concentration. These observations demonstrate that self-association of malvidin-3-glucoside is more important than copigmentation in young red wine.     

Effect of cyclodextrins on the thermodynamic and kinetic properties of cyanidin-3-O-glucoside

In this study, the effect of α and β-cyclodextrin on cyanidin-3-O-glucoside color was investigated by UV–visible absorption techniques. The equilibrium and kinetic constants of the network of chemical reactions taking place in cyanidin-3-O-glucoside were also studied in water at 25 °C by UV–visible absorption techniques. The results showed that the addition of β-cyclodextrin resulted in the fading of anthocyanin solution, and this fading effect was greater at higher pH. This anti-copigmentation effect is caused by the selective inclusion and stabilization of the anthocyanin colorless forms into the β-cyclodextrin cavity. Oppositely, no changes were observed in the cyanidin-3-O-glucoside absorption spectra with the addition of α-cyclodextrin.Direct pH jump, from thermally equilibrated solutions at pH = 1.0 (flavylium cation, AH⁺), shows three kinetic processes: formation of the base A, hydration reaction to form the hemiketal B and the chalcone cis–trans isomerization (Cc–Ct). The results obtained clearly indicated that the equilibrium and kinetic constants of the network of chemical reactions taking place in cyanidin-3-O-glucoside were affected by the presence of β-cyclodextrin. Molecular inclusion in the β-cyclodextrin cavity resulted in the increase of the isomerization observed rate constant (k_obs) at pH 5.3 and in the increase of the hydration equilibrium constant K_h which is in agreement with the fading of the anthocyanin solution. For the macrocycle α-cyclodextrin, no significant changes were observed on the equilibrium and kinetic constants, which suggests that the inclusion of cyanidin-3-glucoside in the α-cyclodextrin's cavity is not favored.     

Electrochemical behaviour and antioxidant capacity of anthocyanins from Chilean red wine,grape and raspberry

The anthocyanin fractions were extracted from Cabernet Sauvignon red wine, skins of Vitis vinifera grapes and raspberry fruits (Rubus idaeus). In red wine extract, 16 anthocyanins were identified, malvidin-3-O-glucoside being the main anthocyanin, which comprised 53.6% of the total anthocyanin in grape extract. Raspberry extract contained mainly delphinidin-3-O-glucoside and cyanidin-3-O-glucoside. The antioxidant capacity of the extracts was assayed by electrochemical methods. Best resolution of the oxidation peaks for the extracts and diluted wine was obtained by pulse differential voltammetry. The wine diluted 20× presented values of P1 (443 mV) and P2 (676 mV) similar to those corresponding to wine extract, and to the anthocyanin malvidin-3-O-glucoside. The antioxidant capacity of anthocyanins in extracts of wine, grape skin and raspberry fruit was also determined by the Trolox equivalent antioxidant capacity (TEAC) method.     

4-Ethylphenol, 4-ethylguaiacol and 4-ethylcatechol in red wines: Microbial formation,prevention, remediation and overview of analytical approaches

The presence of 4-ethylphenol, 4-ethylguaiacol and 4-ethylcatechol in red wines affect negatively their aroma conferring horsy, barnyard, smoky and medicinal aromatic notes. These volatile phenols formed from free hydroxycinnamic acids and their ethyl esters by Dekkera/Brettanomyces yeasts, can contaminate wines. Their formation can cause serious negative economic impact to the wine industry worldwide as consumers tend to reject these wines. For these reasons various preventive and remedial treatments have been studied. This review summarises the wine microbial volatile phenols formation, preventive measures during winemaking and remedial treatments in finished wines along with their advantages and limitations for dealing with this sensory defect and impact on wine quality. Also it is important to control the levels of volatile phenols in wines using fast and convenient analytical methods namely with a detection limit below their olfactory perception threshold. The analytical methods available for quality control and performance characteristics as well their advantages and disadvantages when dealing with a complex matrix like wine are discussed in detail.     

Brettanomyces susceptibility to antimicrobial agents used in winemaking: in vitro and practical approaches

Brettanomyces bruxellensis is a spoiling yeast responsible for developing off-odors in wine described as “Brett-character.” The objective of this study was to evaluate the antimicrobial activity of four enological compounds against Brettanomyces: potassium metabisulfite (PMB), chitosan, enological tannins and dimethyl dicarbonate. Minimal inhibitory concentrations and minimal biocidal concentrations of the antimicrobial agents were determined, and a comparative study between B. bruxellensis and Saccharomyces cerevisiae was performed under in vitro controlled conditions. All tested compounds showed inhibitory effect on the growth of Brettanomyces. Chitosan and the enological tannins showed selectivity against Brettanomyces, and PMB showed the highest efficacy in concentrations under the currently permitted limits for enological use; consequently, PMB was further evaluated in red wines naturally contaminated by Brettanomyces. Volatile phenol concentrations were determined after long-term storage of the wines treated with PMB. A direct correlation was demonstrated between the concentrations of 4-ethylphenol, 4-ethylguaiacol, 4-propylguaiacol and Brettanomyces populations in the studied wines, and these parameters correlated inversely with the concentrations of PMB employed. This is the first time that 4-propylguaiacol is shown to correlate with Brettanomyces population in wine. It is of enological significance that a concentration of 100 mg/L of total PMB efficiently prevented Brettanomyces growth in the aging red wines of our study and that volatile phenol concentrations were significantly (p < 0.05) higher in those poorly protected wines.