The effect of polysaccharide-degrading wine yeast transformants on the efficiency of wine processing and wine flavour

Commercial polysaccharase preparations are applied to winemaking to improve wine processing and quality. Expression of polysaccharase-encoding genes in Saccharomyces cerevisiae allows for the recombinant strains to degrade polysaccharides that traditional commercial yeast strains cannot. In this study, we constructed recombinant wine yeast strains that were able to degrade the problem-causing grape polysaccharides, glucan and xylan, by separately integrating the Trichoderma reesei XYN2 xylanase gene construct and the Butyrivibrio fibrisolvens END1 glucanase gene cassette into the genome of the commercial wine yeast strain S. cerevisiae VIN13. These genes were also combined in S. cerevisiae VIN13 under the control of different promoters. The strains that were constructed were compared under winemaking conditions with each other and with a recombinant wine yeast strain expressing the endo-beta-1,4-glucanase gene cassette (END1) from B. fibrisolvens and the endo-beta-1,4-xylanase gene cassette (XYN4) from Aspergillus niger, a recombinant strain expressing the pectate lyase gene cassette (PEL5) from Erwinia chrysanthemi and the polygalacturonase-encoding gene cassette (PEH1) from Erwinia carotovora. Wine was made with the recombinant strains using different grape cultivars. Fermentations with the recombinant VIN13 strains resulted in significant increases in free-flow wine when Ruby Cabernet must was fermented. After 6 months of bottle ageing significant differences in colour intensity and colour stability could be detected in Pinot Noir and Ruby Cabernet wines fermented with different recombinant strains. After this period the volatile composition of Muscat d'Alexandria, Ruby Cabernet and Pinot Noir wines fermented with different recombinant strains also showed significant differences. The Pinot Noir wines were also sensorial evaluated and the tasting panel preferred the wines fermented with the recombinant strains.     

Antioxidant and Antiproliferative Activities of Twenty-Four Vitis vinifera Grapes

Grapes are rich in phytochemicals with many proven health benefits. Phenolic profiles, antioxidant and antiproliferative activities of twenty-four selected Vitis vinifera grape cultivars were investigated in this study. Large ranges of variation were found in these cultivars for the contents of total phenolics (95.3 to 686.5 mg/100 g) and flavonoids (94.7 to 1055 mg/100 g) and antioxidant activities (oxygen radical absorbance capacity 378.7 to 3386.0 mg of Trolox equivalents/100 g and peroxylradical scavenging capacity14.2 to 557 mg of vitamin C equivalents/100 g), cellular antioxidant activities (3.9 to 139.9 µmol of quercetin equivalents/100 g without PBS wash and 1.4 to 95.8 µmol of quercetin equivalents /100 g with PBS wash) and antiproliferative activities (25 to 82% at the concentrations of 100 mg/mL extracts).The total antioxidant activities were significantly correlated with the total phenolics and flavonoids. However, no significant correlations were found between antiproliferative activities and total phenolics or total flavonoids content. Wine grapes and color grapes showed much higher levels of phytochemicals and antioxidant activities than table grapes and green/yellow grapes. Several germplasm accessions with much high contents of phenolics and flavonoids, and total antioxidant activity were identified. These germplasm can be valuable sources of genes for breeding grape cultivars with better nutritional qualities of wine and table grapes in the future.     

Immunodetection of Botrytis-specific Invertase in Infected Grapes

Invertase (β-fructofuranosidase, EC 3.2.1.26) activity was shown to be stimulated in grape berries after infection with Botrytis cinerea . By using organism-specific extraction methods, evidence was found proving that both partners contribute to the increase in activity. Qualitative analysis of the extracts by SDS–PAGE showed a new invertase species in the botrytised material, with a molecular weight similar to that of Botrytis invertase (BIT). A method allowing the preparative isolation of homogeneous invertase from liquid cultures of B. cinerea in only three steps (ethanol precipitation, DEAE anion exchange and hydroxyapatite chromatography) with 50% recovery was developed. BIT appeared to be strongly glycosylated; interestingly, the amount of glycan seemed to vary and had a remarkable influence on the chromatographic behaviour of the enzyme. After chemical deglycosylation with TFMS, the BIT peptide was used for the production of polyclonal antibodies in chickens (anti-BIT-IgY). The antibodies recognised glycosylated as well as deglycosylated BIT, a partial denaturation of the protein being necessary for reaction. This indicates that the deglycosylation procedure had been successful and that the antibodies were in fact directed to the peptide moiety. However, after a short incubation at 70°C, native BIT was detectable by anti-BIT-IgY, while still active. Western blotting with extracts of diseased berries confirmed the fungal origin of the new invertase form. The anti-BIT-IgY proved highly specific, although some cross-reaction with a protein in Monilia laxa extracts occurred. The importance of careful immunogen preparation in the production of specific antibodies, and the potential of BIT as a target molecule for the immunological detection of B. cinerea are discussed.     

Biological Traits of Xylella fastidiosa Strains from Grapes and Almonds

Xylella fastidiosa is a xylem-limited bacterium that causes various diseases, among them Pierce's disease of grapevine (PD) and almond leaf scorch (ALS). PD and ALS have long been considered to be caused by the same strain of this pathogen, but recent genetic studies have revealed differences among X. fastidiosa isolated from these host plants. We tested the hypothesis that ALS is caused by PD and ALS strains in the field and found that both groups of X. fastidiosa caused ALS and overwintered within almonds after mechanical inoculation. Under greenhouse conditions, all isolates caused ALS and all isolates from grapes caused PD. However, isolates belonging to almond genetic groupings did not cause PD in inoculated grapes but systemically infected grapes with lower frequency and populations than those belonging to grape strains. Isolates able to cause both PD and ALS developed 10-fold-higher concentrations of X. fastidiosa in grapes than in almonds. In the laboratory, isolates from grapes overwintered with higher efficiency in grapes than in almonds and isolates from almonds overwintered better in almonds than in grapes. We assigned strains from almonds into groups I and II on the basis of their genetic characteristics, growth on PD3 solid medium, and bacterial populations within inoculated grapevines. Our results show that genetically distinct strains from grapes and almonds differ in population behavior and pathogenicity in grapes and in the ability to grow on two different media.     

Surface Wax Structure in Fresh and Dried Sultana Grapes

The surface wax of the drying grape sultana has been examinedusing the technique of scanning electron microscopy. The waxlayers of both untreated fresh grapes and of dried grapes weresimilar, and consisted of an elaborate series of overlappingplatelets orientated in all directions. By contrast the waxplatelets of grapes that had been dipped in commercial dips(alkali, fatty acid emulsions) known to accelerate drying wereclosely adpressed to the berry surface. The wax platelets ofgrapes which had been emulsion-dipped and then washed with distilledwater were similar in appearance to those of untreated grapes.The reversible change in wax platelet orientation has not beendescribed before and it is suggested that it may affect thedissemination of water from cuticular surfaces.     

Evolution during wine aging of colour and tannin differences induced by wine starters

The ability of threeSaccharomyces cerevisiae strains, with different colour adsorption aptitude, to induce and maintain colour differences in wines obtained from the Calabrian Gaglioppo and Magliocco black grape varieties was studied during one year of aging. The evolution of wine tannin content was also considered. Total polyphenols, colour parameters and total tannin values exhibited, both for Gaglioppo and Magliocco wines, significant (P < 0.05) or highly significant (P < 0.01) differences among strains. It is interesting to note that yeasts appear to exhibit a different adsorption aptitude for anthocyanins and tannins. The strain that gave wine with high values for the colour parameters was not the same as the one that produced wine with high values of tannins. The obtained results suggest that the choice of yeast strain in winemaking affects, in a significant way, the phenolic composition of wines with direct consequences on their colour and tannin content. Moreover, the interaction between grape cultivar and yeast is close and important, because grape variety, due to its phenolic composition, modulates yeast strain adsorption activity.     

Investigation of Volatile Components in Wines from Koshu and Zenkoji Grapes

A 3-hydroxyethyl-4-cyanoazetidin-2-one derivative (2) was synthesized from (2R,3R)-potassium 2,3-epoxybutyrate through two steps, and it was then further converted to a diazo derivative (7).     

The Grapes checkpoint coordinates nuclear envelope breakdown and chromosome condensation

Mutations in the embryonic Drosophila Grapes/Chk1 checkpoint result in an abbreviated interphase, chromosome condensation defects and metaphase delays. To clarify the relationship between these phenotypes, we simultaneously timed multiple nuclear and cytoplasmic events in mutant grp-derived embryos. These studies support a model in which grp disrupts an S-phase checkpoint, which results in progression into metaphase with incompletely replicated chromosomes. We also show that chromosome condensation is independent of the state of DNA replication in the early embryo. Therefore, grp condensation defects are not a direct consequence of entering metaphase with incompletely replicated chromosomes. Rather, initiation of chromosome condensation (ICC) occurs at the normal time in grp-derived embryos, but the shortened interval between ICC and metaphase does not provide sufficient time to complete condensation. Our results suggest that these condensation defects, rather than incomplete DNA replication, are responsible for the extensive metaphase delays observed in grp-derived embryos. This analysis provides an example of how the loss of a checkpoint can disrupt the timing of multiple events not directly monitored by that checkpoint. These results are likely to apply to vertebrate cells and suggest new strategies for destroying checkpoint-compromised cancer cells.     

Astilbin engeletin in grapes and wine

Astilbin (dihydroquercetin 3-rhamnoside) and engeletin (dihydrokaempferol 3-rhamnoside) were isolated for the first time from grapes. Details of their identification include nonderivatized ¹H NMR spectra. These flavanonol glycosides were concentrated in the skins of white grapes, and were also present in white wines as shown by HPLC. Amounts and relative amounts differed by cultivar. They may be involved in certain discoloration problems during wine processing.     

Studies on Storage of Harvested Grapes for Wine Making

When harvested grape clusters were stored at 40°C, the decrement of acidity in grape juice was recognized. The decrement was caused by transformation of malic acid to lactic acid. It is advised to keep the grapes at 40°C as a storing temperature because of drying efficiency, sterile effect on wild microbes and preservation of biological activity of grape clusters. As the period of storage 4 days for Kôshû and 3 days for Merlot were most suitable.     

Alcohol, wine and platelet function

Epidemiological studies have demonstrated an inverse correlation between moderate wine and alcohol consumption and morbidity and mortality from coronary heart disease. The protective effect has been associated with an increase in the plasma level of HDL cholesterol, as it is well recognized that plasma HDL is inversely correlated with CHD. In addition, it has become evident that blood platelets contribute to the rate of development of atherosclerosis and CHD through several mechanisms. In recent studies it has been shown that the level of HDL cholesterol can explain only 50% of the protective effect of alcoholic beverages; the other 50% may be partly related to a decrease in platelet activity. This anti-platelet activity of wine is explained by ethanol but also by the polyphenolic components with which red wines are richly endowed. Several studies carried out on humans and animals have shown that wine phenolics could exert their effects by reducing prostanoid synthesis from arachidonate. In addition, it has been suggested that wine phenolics could reduce platelet activity mediated by nitric oxide. Moreover, wine phenolics increase vitamin E levels while decreasing the oxidation of platelets submitted to oxidative stress. However, a rebound phenomenon of hyperaggregability is observed after an acute alcohol consumption which is not observed with wine consumption. This protection afforded by wine has been duplicated in animals with grape phenolics added to alcohol. The rebound phenomenon may explain ischemic strokes or sudden deaths known to occur after episodes of drunkenness. It appears that wine, and wine phenolics in particular, could have a more significant inhibitory effect on platelet aggregation and could explain, in part, the hypothesis that red wine is more protective against atherosclerosis and coronary heart disease.     

Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine

The most important factors affecting microbial growth in an alcoholic beverage are the ethanol content and the low pH. The effectiveness of different organic acids in conjunction with ethanol concentration in controlling growth of yeasts was determined for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Brettanomyces lambicus, Pichia anomala, Zygosaccharomyces bailii, Saccharomycodes ludwigii and Kluyveromyces thermotolerans in malt extract broth (MEB). The results are summarized as undissociated concentrations of different acids required to inhibit growth of yeasts in MEB containing 10% (v/v) ethanol. About half the amount of undissociated malic or tartaric acid is necessary for inhibition of the yeasts, compared with acetic and lactic acid but the concentrations of acid necessary to inhibit growth were generally very high and unrealistic in wines for controlling growth of most of the yeasts tested. All the yeasts tested were able to grow in acidified non- or low alcoholic beverages but, at higher ethanol concentrations, Sacch. cerevisiae and Zygosacch. bailii have the greatest spoilage potential.     

Homothallism in wine yeasts

In an attempt to improve by hybridisation strains of pure-culture wine yeasts it could be shown, that of the seven strains used five are homothallic. Evidence is presented suggesting that the remainder are also homothallic.This investigation was aided by New Zealand U.G.C. Grant No. 71/60.     

Demystifying wine expertise: olfactory threshold,perceptual skill and semantic memory in expert and novice wine judges

We investigated recognition and identification of wine-relevant odours as a function of domain-specific expertise. Eleven wine experts and 11 wine novices participated in tasks measuring olfactory threshold, odour recognition, odour identification, and consistency of odour naming. Twenty-four wine-relevant odorants were sampled orthonasally by each participant in the semantic (identification; consistency of naming) and episodic (recognition) memory tasks. Results showed superior olfactory recognition by expert wine judges, despite their olfactory sensitivity and bias measures being similar to those of novices. Contrary to predictions based on reports of an association between odour memory and semantic processing, wine experts did not perform better than novices on the verbal memory tasks. Further, ability to recognize odours and ability to name odours were not positively correlated, although the novices' data showed a trend in this direction. The results imply that the source of superior odour recognition in wine experts was not enhanced semantic memory and linguistic capabilities for wine-relevant odours. One interpretation of the data is that wine experts were less susceptible than wine novices to verbal overshadowing. When forced to identify the odorants, experts' superior perceptual skills protected them from verbal interference, whereas novices' generated verbal representations of the odours were emphasized at the expense of the odorant itself. This has implications for training in wine-evaluation skills.