Effect of ammonium concentration on alcoholic fermentation kinetics by wine yeasts for high sugar content

Kinetics of alcoholic fermentation by Saccharomyces cerevisiae wine strains in a synthetic medium with high sugar content were established for different nitrogen initial content and are presented for four strains. The composition of the medium was close to grape must except that the nitrogen source consisted mainly in ammonium and was varied from 120 to 290 mg N/l assimilable nitrogen. The overall nitrogen consumed was also estimated in order to determine nitrogen requirement variability. The effect of assimilable nitrogen was in general greater on sugar consumption rates than on growth and three kinds of effect on sugar consumption rates were observed: (i) existence of an optimal initial nitrogen level for a maximal sugar consumption rate (inhibition if excess), (ii) no effect of nitrogen beyond the intermediary level (saturation), (iii) sugar consumption rate proportional to the initial nitrogen level (activation). In all cases, the amount of consumed nitrogen increased with its initial concentration and so did the fructophilic capacity of the strains. The optimal requirement varied from 0.62 to 0.91 mg N/g of sugars according to different strains. There was no general correlation between the sugar assimilation rates and the nitrogen requirement.     

Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d'Abruzzo red wine quality

The growth of selected, indigenous Saccharomyces cerevisiae added as starters (SRS1, MS72 and RT73) was monitored during Montepulciano d'Abruzzo wine production. In all the fermentations the addition of the starter, caused a decrease of the non-Saccharomyces yeasts. When strains MS72 and RT73 were used as starters they were detected in the first phases of fermentations, while strain SRS1 competed successfully with native yeasts during all the process. Wines obtained by fermentation with the indigenous starters showed some different characteristics, according to the chemical and sensory analyses. This study highlighted that among selected starters with high fermentative capacity, some are able to dominate better than other natural wine yeast biota, whereas some strains can interact and survive besides native yeast populations during the fermentation. As a consequence, the dominance character can have a positive or negative effect on wine quality and has to be considered in the frame of yeast selection in order to improve or characterize traditional wines. Winemakers could choose among different degrees of yeast dominance to modulate the interaction among starter and native wine yeast population.     

Differential stilbene induction susceptibility of seven red wine grape varieties upon post-harvest UV-C irradiation

The stilbene induction capacity of the red wine grape varieties 'Tempranillo' (TEM), 'Cabernet Sauvignon' (CAS), 'Merlot' (MER), 'Syrah' (SYR), 'Monastrell' (MON), 'Garnacha' (GAR) and 'Cariñena' (CAR) exposed to post-harvest UV-C irradiation is reported for the first time. The induction of trans-piceatannol, trans-resveratrol and viniferins, among other stilbenes, was characterized using high-performance liquid chromatograph with diode-array and mass spectrometric detection (HPLC-DAD-MS-MS). All the wine grape varieties increased their resveratrol, piceatannol and viniferin content after UV-C treatment with the exception of the variety MON in which only piceatannol was induced. Other stilbenes such as astringin (piceatannol glucoside) or piceid (resveratrol glucoside) were induced to a lesser extent and only in some varieties. The stilbene induction capacity of grapes submitted to UV-C irradiation did not necessarily match the final corresponding maximum stilbene content reached in each variety. The total stilbene induction capacity of the grapes ranged from 2.4-fold (in MER) to 10.9-fold (in both CAS and CAR) with respect to untreated grapes (control). The total stilbene content (mg stilbene/100 g fresh weight, fw) after UV-C irradiation ranged from 1.16 in MON to 3.5 in MER. In general, the maximum induction of specific stilbenes in all the varieties followed the sequence: resveratrol >piceatannol >₁-viniferin >piceid. This maximum induction was 22.7-fold for resveratrol in CAS, 6.4-fold for piceatannol in CAR, 3.8-fold for piceid in TEM and 8.4-fold for ₁-viniferin in both CAS and CAR with respect to the control. The maximum concentration of the specific stilbenes reached after UV-C irradiation was 2.5 (mg stilbene/100 g fresh grape weight) for resveratrol in MER, 0.42 for piceatannol in MER, 0.16 for piceid in MON and 0.39 for ₁-viniferin in CAR. The screening of stilbene induction capacity in red wine grapes treated with UV-C irradiation could be useful for selecting the most suitable variety to produce stilbene-enriched red wines with potentially increased health-promoting properties.     

Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.     

Redox effect on volatile compound formation in wine during fermentation by Saccharomyces cerevisiae

Although redox state is a well-known key process parameter in microbial activity, its impact on wine volatile aroma compounds produced during fermentation has not been studied in detail. In this study we report the effect of reductive and microaerobic conditions on wine aroma compound production using different initial amounts of yeast assimilable nitrogen (YAN: 180 and 400 mg N/l) in a simil grape must defined medium and two S. cerevisiae strains commonly used in wine-making. In batch fermentation culture conditions, reductive conditions were obtained using flasks plugged with Muller valves filled with sulphuric acid; while microaerobic conditions were attained with defined cotton plugs. It was found that significant differences in redox potential were obtained using the different plugs, and with variation of over 100 mV during the main fermentation period.     

Ester synthesis and hydrolysis in an aqueous environment,and strain specific changes during malolactic fermentation in wine with Oenococcus oeni

Previous work has shown that Oenococcus oeni produces esterases that are capable of hydrolysing artificial substrates. Using SPME–GCMS, this study provides evidence that purified O. oeni esterases have the ability to both synthesise and hydrolyse esters. Two purified esterases (EstA2 and EstB28) synthesised ethyl butanoate and ethyl hexanoate to varying degrees. Both purified esterases hydrolysed ethyl butanoate, ethyl hexanoate and ethyl octanoate. Once this dual activity was confirmed, malolactic fermentation (MLF) trials were conducted in wine with O. oeni strains that had been previously observed to have either high or low esterase activity against artificial substrates. Strain specific differences were observed and strains with low esterase hydrolysis activity against artificial substrates had a higher level of total esters measured after MLF. The results demonstrate the impact that O. oeni has on wine aroma and relates this to the ester hydrolysis and synthesis abilities of O. oeni strains.     

Nitrogen requirements of commercial wine yeast strains during fermentation of a synthetic grape must

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Currently, the most common method for dealing with nitrogen-deficient fermentations is adding supplementary nitrogen (usually ammonium phosphate). However, it is important to know the specific nitrogen requirement of each strain, to avoid excessive addition that can lead to microbial instability and ethyl carbamate accumulation. In this study, we aimed to determine the effect of increasing nitrogen concentrations of three different nitrogen sources on growth and fermentation performance in four industrial wine yeast strains. This task was carried out using statistical modeling techniques. The strains PDM and RVA showed higher growth-rate and maximum population size and consumed nitrogen much more quickly than strains ARM and TTA. Likewise, the strains PDM and RVA were also the greatest nitrogen demanders. Thus, we can conclude that these differences in nitrogen demand positively correlated with higher growth rate and higher nitrogen uptake rate. The most direct effect of employing an adequate nitrogen concentration is the increase in biomass, which involves a higher fermentation rate. However, the impact of nitrogen on fermentation rate is not exclusively due to the increase in biomass because the strain TTA, which showed the worst growth behavior, had the best fermentation activity. Some strains may adapt a strategy whereby fewer cells with higher metabolic activity are produced. Regarding the nitrogen source used, all the strains showed the better and worse fermentation performance with arginine and ammonium, respectively.     

Tannin profiles of Vitis vinifera L. cv. red grapes growing in Lisbon and from their monovarietal wines

The tannin profiles of five grape (Vitis vinifera L.) varieties ‘Touriga Nacional’, ‘Trincadeira’, ‘Castelão’, ‘Syrah’ and ‘Cabernet Sauvignon’ and the profiles of their red monovarietal wines from their 2004 and 2005 vintages were studied. Depending on the variety, the polymeric fractions represented 77–85% in seeds and 91–99% in skins. The distribution of the mean degree of polymerisation (mDP) of the proanthocyanidins ranged from 2.8 to 12.8 for seeds and from 3.8 to 81.0 for skins. In the monovarietal wines, the distribution of the mDP of the proanthocyanidins ranged from 2.1 to 9.6. Of the total proanthocyanidins the polymeric fraction represented 77–91% in vintage 2004 and 82–95% in vintage 2005. The wine proanthocyanidins of Trincadeira and Cabernet Sauvignon showed similar tannin profiles in each vintage. After six months of storage, noticeable decreases in total proanthocyanidins concentration were measured. These were accompanied by slight decreases in prodelphinidin percentages but the percentage of galloylation and mDP remained similar.     

Extraction,distribution and characterisation of phenolic compounds and oil in grapeseeds

Five factors, extraction means, extraction repeats, extraction solvents, sample granularities, and grinding manners, were investigated and compared to optimise the extraction methods of phenolic compounds from grapeseed. Twenty minutes sequential sonication for three repeats was a preferred extraction means for assessment of phenolic content in grapeseed over three repeats 100-min sequential mechanical agitation. Methanol/water/HCl (70:29:1, v/v/v) was the best among the solvent compositions tested. Grinding granularity showed a significant (p = 0.05) effect on the yield of phenolic compounds when the sample was ground with a grinder and separated to different granularities by sieves. Grinding sample with mortar in liquid nitrogen could provide samples with uniform size and thus give a more consistent evaluation of phenolic compounds in grapeseed. Significant correlation coefficients (p < 0.01) among total phenols, total flavan-3-ols, and total proanthocyanidins contents in grapeseed extracts were observed. In addition, phenolic compounds and oil were very unevenly distributed in grapeseed as about 90% of phenolic compounds were observed in seed coat while about 60% of oil was in the endosperm and embryo.     

Effect of the addition of different quantities of amino acids to nitrogen-deficient must on the formation of esters, alcohols, and acids during wine alcoholic fermentation

In this work the effect of the addition of different quantities of amino acids to a nitrogen-deficient must on the formation of volatile compounds during the wine alcoholic fermentation was studied. To do so, fermentations of Mazuelo must were carried out, to which were added ammonium and 45, 120, and 250 mg/l of amino acids. The results showed that the formation of total esters, isoamyl acetate, and 2-phenylethyl acetate was directly proportional to the quantity of amino acids added to the must while the synthesis of diethyl succinate, and ethyl 3-hydroxybutyrate was inversely proportional to this addition. The alcohols, with the exception of tyrosol and 2-phenylethanol, did not show any direct correlation between their formation and the addition of amino acids. The quantity of amino acids added to the must favoured the formation of total acids but it had scant influence on the individual synthesis of the acids. So, it may be stated that, in general, the addition of amino acids in the concentrations under study to a nitrogen-deficient must, favoured the formation of volatile compounds in the wine.     

Effect of aromatic precursor addition to wine fermentations carried out with different Saccharomyces species and their hybrids

This work explores the ability of different yeast strains from different species of the genus Saccharomyces (S. cerevisiae, S. uvarum and S. kudriavzevii) and hybrids between these species to release or form varietal aroma compounds from fractions of grape odourless precursors. The de novo synthesis by the yeasts of some of the varietal aroma compounds was also evaluated. The study has shown that de novo synthesis affects some lipid derivatives, shikimic derivatives and terpenes in all species and hybrids, with some remarkable differences amongst them. The release or formation of aroma compounds from precursors was found to be strongly linked to the yeast or hybrid used, and the triple hybrid S. cerevisiae × S. bayanus × S. kudriavzevii in particular and secondarily the hybrid S. cerevisiae × S. bayanus were highly efficient in the production of most varietal aroma compounds, including γ-lactones, benzenoids, volatile phenols, vanillin derivatives and terpenols. The presence of precursors in the fermenting media caused a surprising levelling effect on the fermentative aroma composition. Altogether, these results suggest that it is possible to modulate wine aroma by employing different yeast species in order to create new wines with different aromatic notes.     

Effect of sarsaponin on ruminal fermentation with particular reference to methane production in vitro

This experiment was designed to investigate the effects of different concentrations (0, 1.2, 1.8, 2.4, and 3.2 g/L) of sarsaponin on ruminal microbial methane production using the substrates soluble potato starch, cornstarch, or hay plus concentrate (1.5:1). Ruminal fluid was collected from a dairy cow, mixed with phosphate buffer (1:2) and incubated (30 ml) anaerobically at 38 degrees C for 6 and 24 h with or without sarsaponin. Excluding the lower level of sarsaponin, pH of the medium was slightly decreased. Ammonia-N concentration and numbers of protozoa were decreased in a dose-dependent manner. Total volatile fatty acids and total gas production were increased. Molar proportion of acetate was decreased and propionate was increased with a corresponding decrease in acetate:propionate ratio. Hydrogen production was decreased. As the concentration of sarsaponin increased from 1.2 to 3.2 g/L, fermentation of soluble potato starch, cornstarch, or hay plus concentrate decreased methane production from 20 to 60% (6 h) and 17 to 50% (24 h), 21 to 58% (6 h) and 18 to 52% (24 h), and 23 to 53% (6 h) and 15 to 44% (24 h), respectively. Excluding the lower dose concentration (1.2 g/L) of sarsaponin, in vitro disappearance of dry matter of hay plus concentrate was decreased after 24 h. In conclusion, these results show that sarsaponin stimulated the mixed ruminal microorganism fermentation as well as to inhibit methane production in vitro.     

Chitosan elicits mono-glucosylated stilbene production and release in fed-batch bioreactor cultures of grape cells

The present paper reports the study of the optimal conditions for grape (Vitis vinifera cv. Barbera) cell suspensions in batch and fed-batch bioreactor cultures, in order to specifically improve the production of mono-glucosylated stilbenes, which are resveratrol derivatives. These compounds are physiologically as active as free resveratrol in cardio- and chemoprotection, but are more stable and bioavailable after ingestion through diet. In fed-batch conditions the production of mono-glucosides was considerably increased together with that of free resveratrol. For the first time, an elicitor (chitosan) was tested in a bioreactor system, demonstrating its efficacy in inducing the production of stilbenes. The bioreactor culture conditions also allowed the accumulation of other polyphenols, such as catechins. The vast majority of the produced compounds was released into the culture media, which represents a relevant advantage for the recovery of specific molecules or of polyphenol-enriched mixtures. These results represent a further step toward the employment of grape cells in fed-batch cultures, as a promising alternative to whole plant extraction for the industrial production of plant polyphenols, considering the necessity for developing sustainable processes.     

Influence of extraction methodology on grape composition values

This work demonstrated similarities and differences in quantifying many grape quality components (>45 compounds) that were extracted from berries by three distinct preparations, before being analysed by eight spectrophotometric and HPLC methods. All sample extraction methods were appropriate for qualitative results only. Different extraction procedures showed altered component composition in ‘Pinot noir’ berries, possibly due to the localisation of the compounds of interest within the grape and how those compounds were extracted from the berry. Sample extraction is an often-overlooked part of berry evaluations, but this study illustrates that it should be carefully considered prior to berry component analysis for its influence upon measurements.     

The production of ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera: A review

This work reviews the formation of ethylphenols (4-ethylphenol and 4-ethylguaiacol) from grape hydroxycinnamic acids in aging red wines by yeasts of the genera Brettanomyces and Dekkera. The physico-chemical factors that favour the growth of these undesirable yeasts, the techniques used to detect the presence of Brettanomyces/Dekkera species in wines, and the analytical techniques for monitoring the formation of volatile ethylphenols are all described. Finally, the advantages and disadvantages of the different options for controlling the growth of these yeasts are discussed.     

Effect of phenolic acids on glucose and organic acid metabolism by lactic acid bacteria from wine

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.     

Effect of oxygen and lipid supplementation on the volatile composition of chemically defined medium and Chardonnay wine fermented with Saccharomyces cerevisiae

Oxygen or lipids are required to complete stressful alcoholic fermentation. Lack of these nutrients can inhibit sugar uptake and growth, which leads to incomplete or ‘stuck’ fermentation. Oxygen or lipids supplementation not only restores yeast fermentative activity and also affects formation of yeast volatile metabolites. To clarify the effect of oxygen and lipid supplementation on the formation of flavour active metabolites during wine fermentation, we evaluated the addition of these two nutrients to chemically defined grape juice and filter clarified Chardonnay must. Lipid addition increased the concentration of esters, higher alcohols and volatile acids, whereas oxygen increased the concentration of higher alcohols and altered the proportion of acetate to ethyl esters and the proportion of branch-chain acids to medium-chain fatty acids. Combined addition of lipids and oxygen showed an additive effect on concentration of higher alcohols whereas oxygen suppressed the enhancing effect of lipids on formation of esters and volatile acids. Our results demonstrate the potential of lipid and oxygen supplementation for the manipulation of wine aroma in white wine fermentation.     

Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine

In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine.     

Effect of intra and interspecific interaction on OTA production by A. section Nigri in grapes during dehydration

The aim of this study was to assess the colonizing capability and OTA production of different populations of Aspergillus section Nigri spp. in grapes, as affected by the interactions with other fungi, during a simulated in vitro sun-drying. Mature white grapes were divided into two lots of healthy and artificially injured grapes and inoculated with A. section Nigri spp. (A. carbonarius OTA producer, A. niger aggregate OTA producer, A. niger aggregate OTA non-producer), Eurotium amstelodami and Penicillium janthinellum, in different combinations. The drying process was simulated adjusting water activity firstly at 0.98 a(w) and gradually decreasing it to 0.76 a(w) for a total of 20 days. Colonizing grape percentages were recorded after 5, 10, 15 and 20 days of dehydration and OTA content was measured after 5, 7, 10, 12, 15, 17 and 20 days. Colonization of grapes increased with time in all treatments. A. niger aggregate OTA-positive showed the highest colonization percentage, followed by A. carbonarius, and finally their mixed inoculum. When the two OTA-producing strains were combined, addition of any other microorganism increased the percentage of infection by A. section Nigri. A. carbonarius was the highest OTA producer in pure culture, followed by A. niger aggregate OTA-positive. In general, when competing fungi were added to A. carbonarius inoculum, the OTA content was reduced. E. amstelodami was the only competing fungus which increased OTA accumulation. The sun-drying process may be conducive to OTA accumulation in dried grapes. The complex fungal interactions which may take place during this process, may act as an additional control factor, given that the higher presence of A. niger aggregate OTA-negative inhibits OTA accumulation by OTA producing species.     

Polyphenolic profiles detected in the ripe berries of Vitis vinifera germplasm

Polyphenolic profiles in the berry samples of 344 European grape (Vitis vinifera) cultivars were evaluated for two consecutive years. These cultivars represent a diverse collection of V. vinifera germplasm maintained at the USDA-Agricultural Research Service Vitis Clonal Repository in Davis of California, USA. A total of 36 polyphenolic compounds, including 16 anthocyanins, 6 flavonols, 6 flavanols, 6 hydroxycinnamic acids and 2 hydroxybenzoic acids, were identified via HPLC–MS and quantified by HPLC–DAD. The mean contents for anthocyanins, flavanols, flavonols, hydroxycinnamic acids and hydroxybenzoic acids were 0.946 (coloured cultivars), 0.147, 0.043, 0.195 and 0.016 mg g⁻¹ FW, respectively. On average, wine grapes had higher concentrations than had table grapes for all of these compounds except hydroxycinnamic acids. Berry colours affected the total contents of anthocyanins, but not others. Positive correlations (0.151–0.535) were found among these groups of compounds. As expected, these groups of compounds were all negatively correlated with berry weight.