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.     

Influence of Dekkera bruxellensis on the contents of anthocyanins,organic acids and volatile phenols of Dão red wine

The anthocyanin, organic acid and volatile phenol compositions of red wine obtained from Touriga Nacional grapes growing in the Dão region (Portugal) were determined by HPLC/DAD, HPLC/UV and GC/FID, respectively. By these means, nine anthocyanic compounds (malvidin-3,5-O-diglucoside, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin, cyanidin, pelargonidin and malvidin), six organic acids (ketoglutaric, tartaric, malic, quinic, lactic and shikimic acids) and two volatile phenols (4-ethylguaiacol and 4-ethylphenol) were identified and quantified. Malvidin-3-O-glucoside, the pair lactic plus shikimic acids and 4-ethylguaiacol were the main anthocyanin, organic acids and volatile phenol, respectively. The effects of nine different Dekkera bruxellensis strains on these chemical parameters were also evaluated. The results obtained indicate that some strains of D. bruxellensis yeast are able to cause deterioration of red wine from the Dão region during its maturation by the production of volatile phenols, namely 4-ethylphenol.     

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.     

Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines

Eighty four isolates of Brettanomyces bruxellensis, were collected during fermentation of Sangiovese grapes in several Tuscan wineries and characterized by restriction analysis of 5.8S-ITS and species-specific PCR. The isolates were subsequently analysed, at strain level, by the combined use of the RAPD-PCR assay with primer OPA-02 and the mtDNA restriction analysis with the HinfI endonuclease. This approach showed a high degree of polymorphism and allowed to identify seven haplotypes, one of them being the most represented and widely distributed (72 isolates, 85.7%). Physiological traits of the yeasts were investigated under a wine model condition. Haplotypes clustered into two groups according to their growth rates and kinetics of production of 4-ethylphenol and 4-ethylguaiacol. Hexylamine was the biogenic amine most produced (up to 3.92 mg l^− 1), followed by putrescine and phenylethylamine. Formation of octapamine was detected by some haplotypes, for the first time.     

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.     

RNA fingerprinting analysis of Oenococcus oeni strains under wine conditions

Oenococcus oeni is a lactic acid bacterium of economic interest used in winemaking. This bacterium is the preferred species for malolactic fermentation (MLF) due its adaptability to the chemically harsh wine environment. MLF enhances the organoleptic properties and ensures deacidification of wines.     

4-Ethylphenol and 4-ethylguaiacol depletion in wine using esterified cellulose

The ability of cellulose acetate, cellulose acetate propionate (CAP), cellulose acetate butyrate and cellulose propionate (CP) fibres to reduce 4-ethylphenol and 4-ethylguaiacol, causing the off-flavour named “Brett character”, was studied. CAP and CP performed best in a preliminary comparative test. The former was chosen because of its more favourable FDA classification for food contact substances. CAP effectiveness was studied in relation to the reduction in volatile phenols with doses of up to 20 g/l and wine contact times of up to 60 min. Using 4 g/l, reduction of both phenols was, on average, 31–32% in defective red wines. Wine treatment affected neither colour nor total proanthocyanidins and catechins, and wines were judged to be better than the corresponding spoiled controls. CAP fibre can be regenerated by washing with ethanol or aqueous solution (pH 12), without notable changes in depletion efficiency. The technological characteristics of CAP and, to a lesser extent, CP offer interesting prospects for producing filtration beds, pads and membranes for the treatment of wines and beverages affected by “Brett character”.     

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.     

The effect of multiple yeasts co-inoculations on Sauvignon Blanc wine aroma composition,sensory properties and consumer preference

Consumer acceptance testing has been only recently applied in wine research, to assess wine sensory attributes that affect hedonic liking. The aim of this study was to investigate the effect of Saccharomyces yeast co-inoculations on wine volatile composition and sensory profiles, and to determine if differences were sufficient enough to affect consumer acceptance. Fermentations were conducted using two- and three-yeast co-inoculations, and single strains. Yeast inocula differed substantially in volatile thiols and other flavour compounds, and in their sensory properties. Wines from four yeast inocula which showed large sensory differences were subjected to consumer testing by 120 consumers, with differences in overall liking found. Four clusters of consumers were identified, with one group strongly preferring the two-yeast co-inoculated wine with an intermediate sensory profile, while another group favoured the wine made using the three-yeast co-inoculation. This study has demonstrated that the yeast inoculum used to conduct fermentation affects consumer acceptance.     

Fermentative behavior of Saccharomyces strains during microvinification of raspberry juice (Rubus idaeus L.)

Sixteen different strains of Saccharomyces cerevisiae and Saccharomyces bayanus were evaluated in the production of raspberry fruit wine. Raspberry juice sugar concentrations were adjusted to 16°Brix with a sucrose solution, and batch fermentations were performed at 22 °C. Various kinetic parameters, such as the conversion factors of the substrates into ethanol (Y_p/s), biomass (Y_x/s), glycerol (Y_g/s) and acetic acid (Y_ac/s), the volumetric productivity of ethanol (Q_p), the biomass productivity (P_x), and the fermentation efficiency (E_f) were calculated. Volatile compounds (alcohols, ethyl esters, acetates of higher alcohols and volatile fatty acids) were determined by gas chromatography (GC-FID). The highest values for the E_f, Y_p/s, Y_g/s, and Y_x/s parameters were obtained when strains commonly used in the fuel ethanol industry (S. cerevisiae PE-2, BG, SA, CAT-1, and VR-1) were used to ferment raspberry juice. S. cerevisiae strain UFLA FW 15, isolated from fruit, displayed similar results. Twenty-one volatile compounds were identified in raspberry wines. The highest concentrations of total volatile compounds were found in wines produced with S. cerevisiae strains UFLA FW 15 (87,435 μg/L), CAT-1 (80,317.01 μg/L), VR-1 (67,573.99 μg/L) and S. bayanus CBS 1505 (71,660.32 μg/L). The highest concentrations of ethyl esters were 454.33 μg/L, 440.33 μg/L and 438 μg/L for S. cerevisiae strains UFLA FW 15, VR-1 and BG, respectively. Similar to concentrations of ethyl esters, the highest concentrations of acetates (1927.67 μg/L) and higher alcohols (83,996.33 μg/L) were produced in raspberry wine from S. cerevisiae UFLA FW 15. The maximum concentration of volatile fatty acids was found in raspberry wine produced by S. cerevisiae strain VR-1. We conclude that S. cerevisiae strain UFLA FW 15 fermented raspberry juice and produced a fruit wine with low concentrations of acids and high concentrations of acetates, higher alcohols and ethyl esters.     

Effect of fusel oil addition on volatile compounds in papaya wine fermented with Williopsis saturnus var. mrakii NCYC 2251

The impact of fusel oil addition on volatile compounds formation in papaya wine fermented with yeast Williopsis saturnus var. mrakii NCYC2251 was studied with a view to enhancing papaya wine aroma production. Time-course papaya juice fermentations were carried out using W. saturnus var. mrakii NCYC2251 with fusel oil added (0, 0.1 and 0.5% v/v). Fermentation characteristics in terms of yeast growth, Brix and pH changes were similar for all fermentations except for those added with 0.5% (v/v) fusel oil. The addition of 0.5% (v/v) fusel oil inhibited yeast growth. A wide range of volatile compounds were produced during fermentation including acids, alcohols, esters and aldehydes with esters being the most abundant volatile compounds produced. The addition of 0.1% (v/v) fusel oil reduced the production of undesirable volatiles such as ethyl acetate and acetic acid, while increasing the desirable volatiles production such as ethanol and acetate esters. This study suggests that papaya juice fermentation with W. saturnus var mrakii NCYC 2251 together with a low concentration of added fusel oil can be another means of modulating papaya wine aroma compound formation.     

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.     

Evaluation of the inhibitory effect of dimethyl dicarbonate (DMDC) against wine microorganisms

Several microbial species associated with wine were challenged against increasing concentrations of dimethyl dicarbonate (DMDC). The concentration inducing complete cell death upon addition to red wine was regarded as the minimum inhibitory concentration (MIC). In dry red wines with 12% (v/v) ethanol and pH 3.50, the inactivation depended on the initial cell concentration. For an initial inoculum of 500 CFU/ml, the MIC of the yeasts species Schizosaccharomyces pombe, Dekkera bruxellensis, Saccharomyces cerevisiae and Pichia guilliermondii was 100mg/l. The most sensitive strains belong to Zygosaccharomyces bailii, Zygoascus hellenicus and Lachancea thermotolerans, with MIC of 25mg/l DMDC. For inoculation rates of about 10(6)CFU/ml, the maximum dose of DMDC legally authorized (200mg/l) was not effective against the most resistant species. The addition of 100mg/l potassium metabisulphite (PMB), equivalent to 1mg/l molecular sulphur dioxide, increased the inactivation effect of 100mg/l DMDC over initial yeast populations of 10(6)CFU/ml but did not fully kill S. pombe and S. cerevisiae. Lactic acid and acetic acid bacteria were not killed by the addition of 300 mg/l of DMDC. Trials performed in wines before bottling showed that in most samples indigenous bacterial populations were not affected by 200mg/l DMDC. Therefore, under winery practice, DMDC at the maximum dose legally permitted may be regarded as an efficient preservative to control low contamination rates of yeasts but ineffective against lactic acid and acetic acid bacteria.     

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.     

Ability of Lactobacillus brevis strains to degrade food phenolic acids

The potential to degrade 15 food phenolic acids was investigated for several Lactobacillus brevis strains isolated from different sources. All the strains analysed in this study showed a similar metabolism on phenolic acids. Among the cinnamic acids assayed, only p-coumaric, ferulic and caffeic acids were metabolized by the L. brevis strains. These acids were decarboxylated to produce their corresponding vinyl derivatives. Contrarily to the results previously reported on Lactobacillus plantarum, the L. brevis strains analysed in this study were unable to subsequently reduce or metabolize these vinyl derivatives. In L. brevis, vinyl phenol, vinyl catechol, and vinyl guaiacol were the final metabolic products from p-coumaric, caffeic or ferulic acids, respectively. From the benzoic acids analysed, and similarly to L. plantarum strains, only gallic and protocatechuic acids were modified by L. brevis strains. Both acids were decarboxylated to pyrogallol and catechol, respectively. Currently, the enzymes involved in the metabolism of phenolic acids in L. brevis remain uncharacterized.     

Polyphenols and antioxidant capacity of Phaseolus vulgaris stored under extreme conditions and processed

Phaseolus vulgaris is a foodstuff that not only supplies nutrients, but also bioactive compounds with antioxidant capacity; however, bad post-harvest storage or processing can diminish the antioxidant capacity. In this work, the effect of storage under extreme conditions, of thermal processing and of fermentation on the content of phenolic compounds and their antioxidant capacity (AA) in beans of just harvested P. vulgaris were evaluated. Combination of temperatures of 30 and 50 °C and relative humidity of 11% and 80% for 150 days were evaluated. Extreme storage conditions drastically decreased the AA, demonstrating that post-harvest storage at low temperature and humidity preserves the antioxidant capacity of the beans. Regarding processing, cooking decreased the scavenging effect and the AA by 63.5% and 56.8%, while fermentation decreased them by only 14% and 22%, respectively. It was concluded that the loss of antioxidant capacity due to heating is less in fermented beans.     

Impact of Debaryomyces hansenii strains inoculation on the quality of slow dry-cured fermented sausages

Debaryomyces hansenii strains, M4 and P2, isolated from natural fermented sausages were inoculated in slow fermented sausages to study their effect on processing parameters, microbial population, volatile compound and sensory characteristics. The inoculation of D. hansenii strains, M4 and P2, did not affect the ripening process as no differences in pH and Aw were detected. The dominance of the inoculated yeast strains along the process was followed by RAPDs of M13 minisatellite. The inoculated yeasts, P2 and M4, were recovered at the end of the ripening process although P2 appeared in higher counts than M4. The sausages inoculated with P2 resulted in a decrease in lipid oxidation values (TBARS) and a reduction of lipid-oxidation derived aldehydes in addition to a highest acid compound abundance. M4 inoculated sausages resulted in highest sulphur containing compound abundance. However, no differences in consumer acceptance were detected. Moreover, both yeast strains were responsible for the generation of ethyl methyl-branched ester compounds in the dry-cured sausages.     

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.     

Aromatic series in sherry wines with gluconic acid subjected to different biological aging conditions by Saccharomyces cerevisiae var. capensis

Wines from healthy grapes supplemented with gluconic acid were subjected to biological aging under two experimental conditions. The first one was carried out under flor yeast velum as in the traditional biological aging and the second one under submerged cultures. The highest gluconic acid consumption was observed in aged wines in submerged cultures. Nine aromatic series were obtained by grouping the 48 volatile compounds quantified in wines. The aroma profile based on the aromatic series allows comparison of the changes due to the gluconic acid consumption and the changes due to the different aging conditions assayed. Only the herbaceous and fatty series showed diminished values of consequence of gluconic acid consumption. The fatty, herbaceous and roasty series show highest values, whereas the fruity, floral, solvent and medicinal series reached lower values in the submerged cultures assay. The application of the assay conditions to winemaking can reduce the gluconic acid concentration in wines obtained from rotten grapes.     

Characteristics of some traditional Vietnamese starch-based rice wine fermentation starters (men)

In the Mekong Delta region of South-Vietnam, wine from purple glutinous rice is particularly interesting because of its sherry-like taste and flavour and its attractive brown-red colour. It is manufactured at home or by small cottage industries, using traditional solid-state starters (Men). With the objective of improving the knowledge about the functionality of traditional Men, this study deals with the properties and composition of 29 samples of Vietnamese commercial rice wine starters. We selected 6 rice wine starters for their superior ability to liquefy cooked rice, high ethanol accumulation, and production of attractive flavour and colour in the resulting wine. Ethanol contents reached 12 g/100 ml, a sweet alcoholic fragrance was noticed and the wine colour varied from red to lightly brown. Total mould, yeast and bacteria counts in Men were 3.4-6.0, 5.8-7.2 and 2.6-6.2 log CFU/g of dry weight sample, respectively. A total of 119 microbial strains, comprising 53 moulds, 51 yeasts and 15 bacteria, was isolated. Mould isolates with excellent functionality were identified as Amylomyces rouxii, Amylomyces aff. rouxii (an atypical form of A. rouxii), Rhizopus oligosporus and Rhizopus oryzae. Yeast isolates with excellent fermentation properties were all identified as Saccharomyces cerevisiae; other, less functional isolates were identified as Candida glabrata and Pichia anomala.