Evaluation of sequential inoculation of Saccharomyces cerevisiae and Oenococcus oeni strains on the chemical and aromatic profiles of cherry wines

The current study was carried out to elucidate the effect of sequential inoculation of Saccharomyces cerevisiae (RC212, D254) and Oenococcus oeni (SG26, Lalvin 31 and Uvaferm Alpha) on the production of cherry wines, especially on the chemical and aromatic characteristics. SI-D culture required the shortest period (23 d) to complete the fermentation, while other inoculations needed longer time. Analysis from chemical composition showed that titratable acidity and content of l-malic acid exhibited evident differences among the samples after MLF. For volatile compounds, 49 major components were identified, mostly comprising of alcohols, acids and esters. Cherry wines obtained from SI-B and SI-C showed higher contents of total volatile alcohols, and SI-D wines produced the greatest amount of volatile acids. According to the odour active value (OAV), 9 out of 49 studied volatile components had OAV >1 in all the analyzed wines, while six volatile components showed OAV >1 only for some of them. Furthermore, a sensory analysis was performed to compare the sensory profile of these cherry wines, and results evidenced that wines resulting from different inoculations presented diverse sensory profiles. These findings suggest that sequential inoculations posed a great potential in affecting and modulating the aromatic profile of cherry wines.     

Evaluation of different Saccharomyces cerevisiae strains on the profile of volatile compounds and polyphenols in cherry wines

Tart cherries of ‘Early Richmond’, widely grown in Shandong (China), were fermented with six different Saccharomyces cerevisiae strains (BM4×4, RA17, RC212, D254, D21 and GRE) to elucidate their influence on the production of volatiles and polyphenols. Acetic acid and 3-methylbutanol were found in the highest concentrations among all identified volatiles with all six yeast strains, followed by 2-methylpropanol and ethyl lactate. RA17 and GRE cherry wines were characterised by a higher amount of esters and acids. D254 wine contained a higher concentration of alcohols. With respect to polyphenols, five phenolic acids and four anthocyanins were identified among all tested samples, with chlorogenic and neochlorogenic acids, cyanidin 3-glucosylrutinoside and cyanidin 3-rutinoside being the major compounds. When using principal component analysis to classify the cherry wines according to the volatiles and polyphenols, they were divided into three groups: (1) RA17 and GRE, (2) RC212 and D254 and (3) BM4×4 and D21.     

Multifactorial analysis of acetaldehyde kinetics during alcoholic fermentation by Saccharomyces cerevisiae

Acetaldehyde is the terminal electron acceptor in the alcoholic fermentation by Saccharomyces cerevisiae. Quantitatively the most important carbonyl by-product, it has relevance for ethanol production yields as well as product stabilization and toxicology. The aim of this study was to investigate the effect of various enological parameters on acetaldehyde kinetics during alcoholic fermentations. Two commercial yeast strains were tested in two grape musts and the pH, temperature, SO₂ and nutrient addition were varied. All incubations had uniform kinetics where acetaldehyde reached an initial peak value followed by partial reutilization. Peak acetaldehyde concentrations and residual concentrations after 15 days of fermentations ranged from 62 to 119 mg l^− 1 and 22 to 49 mg l^− 1, respectively. A positive linear relationship was found between peak and final acetaldehyde levels in Gewürztraminer, but not Sauvignon Blanc fermentations, where sluggish fermentations were observed. Several factors had a significant effect on peak and/or final acetaldehyde levels. SO₂ addition, grape cultivar and fermentation nutrition were important regulators of peak acetaldehyde production, while final acetaldehyde concentrations were correlated with SO₂ addition, grape cultivar and temperature. The results allowed to estimate the acetaldehyde increase caused by SO₂ addition to 366 ??g of acetaldehyde per mg of SO₂ added to the must. The course of the final fermentation phase was shown to determine acetaldehyde residues. Comparison of acetaldehyde and hexose kinetics revealed a possible relationship between the time of occurrence of peak acetaldehyde concentrations and the divergence of glucose and fructose degradation rates.     

Acetic acid removal by Saccharomyces cerevisiae during fermentation in oenological conditions. Metabolic consequences

The commercial Saccharomyces cerevisiae strains used in champagne winemaking were tested for their ability to metabolise acetic acid during alcoholic fermentation. Fermentation tests were performed in conditions close to oenological ones using a Chardonnay grape juice supplemented with acetic acid. The amount of acetic acid metabolised by wine yeast increased with increasing initial acetic acid concentration and this elimination occurred during the second part of the exponential growth phase. When the initial acetic acid concentration exceeds 1 g/l, and whatever the yeast strain used, the concentration of acetic acid in the resulting wine cannot be reduced to an acceptable level according to the current legislation. Acetic acid removal modified yeast metabolism, since more acetaldehyde, less glycerol and less succinic acid were produced. Considering the reduction of the NADPH/NADP⁺ ratio following acetic acid consumption, we propose, as a new hypothesis, that acetic acid could modify yeast metabolism by reducing the activity of the NADP⁺ dependent aldehyde dehydrogenase Ald6p.     

High-cell-density fermentation of Saccharomyces cerevisiae for the optimisation of mead production

Mead is a traditional drink that contains 8%–18% (v/v) of ethanol, resulting from the alcoholic fermentation of diluted honey by yeasts. Mead fermentation is a time-consuming process and the quality of the final product is highly variable. Therefore, the present investigation had two main objectives: first, to determine the adequate inoculum size of two commercial wine-making strains of Saccharomyces cerevisiae for the optimisation of mead fermentation; and second, to determine if an increase in yeast pitching rates in batch fermentations altered the resulting aroma profiles. Minor differences were detected in the growth kinetics between the two strains at the lowest pitching rate. With increasing pitching rates net growth of the strain ICV D47 progressively decreased, whereas for the QA23 the increasing inoculum size had no influence on its net growth. The time required to reach the same stage of fermentation ranged from 24 to 96 h depending on the inoculum size. The final aroma composition was dependent on the yeast strain and inoculum size. Fourteen of the twenty-seven volatile compounds quantified could contribute to mead aroma and flavour because their concentrations rose above their respective thresholds. The formation of these compounds was particularly pronounced at low pitching rates, except in mead fermented by strain ICV D47, at 10⁶ CFUs/mL. The esters isoamyl acetate, ethyl octanoate and ethyl hexanoate were the major powerful odourants found in the meads. The results obtained in this study demonstrate that yeast strain and inoculum size can favourably impact mead's flavour and aroma profiles.     

Formation of vinylphenolic pyranoanthocyanins by Saccharomyces cerevisiae and Pichia guillermondii in red wines produced following different fermentation strategies

The strains of two species of yeast, Saccharomyces cerevisiae and Pichia guillermondii, both with high hydroxycinnamate decarboxylase (HCDC) activity (56% and 90% respectively), were used in the fermentation of musts enriched with grape anthocyanins, to favour the formation of highly stable vinylphenolic pyranoanthocyanin pigments. The different strains were used to ferment the must separately, simultaneously, or sequentially, the latter involving an initial period using the yeast with the greatest HCDC activity (P. guillermondii). The must was made from concentrated grape juice diluted to 220 g/l of sugar, and enriched with grape anthocyanins to 100 mg/l and with p-coumaric acid to 120 mg/l. The pH was fixed to 3.5. All 50 ml micro-fermentations were done in triplicate. The development of anthocyanin-3-O-glucoside precursors, the decarboxylation of p-coumaric acid, and the formation of 4-vinylphenol and vinylphenolic pyranoanthocyanin derivatives were studied during the fermentation. The fermentation strategy used and the yeast HCDC activity significantly influenced the formation of vinylphenolic pyranoanthocyanins. The latter molecules were separated, identified, and quantified using high performance liquid chromatograph with diode array detection and electrospray-mass spectrometry (HPLC-DAD-ESI/MS). The volatile compounds profile was screened during fermentation using gas cromatogrphy-flame ionisation detection (GC-FID), in order to detect and quantify the main molecules. The best results were reached with the sequential fermentation (3.74 mg/l of malvidin-3-O-glucoside-4-vinylphenol). This work shows that during mixed or sequential fermentations carried out with non-Saccharomyces or highly fermentative Saccharomyces strains, with high HCDC activity, the content of stable pigments can be increased without sensorial modifications.     

Influence of freeze-drying conditions on survival of Oenococcus oeni for malolactic fermentation

Malolactic fermentation (MLF) is an important process in wine production. Oenococcus oeni is most often responsible for MLF. Starter culture technology, involving the inoculation of O. oeni into wines, has been developed for inducing MLF. In this study, the effects of cell washing, pH of suspension medium, preincubation in sodium glutamate, initial cell concentration and freezing temperature on viability of freeze-dried O. oeni H-2 were investigated. The cell viability of samples without washing with potassium phosphate buffer was significantly lower than those samples undergone washing. When pH of suspension medium was 7.0 the cell survival was the highest. The cell viability was enhanced when the cells were preincubated at 25 °C before freezing. When 2.5% sodium glutamate was used as protective agent in suspension medium, the optimal initial cell concentration was 10⁹ CFU/ml. The cell viability increased by 21.6% as freezing temperature decreased from − 20 °C to − 65 °C. However, when the cells were frozen in liquid nitrogen (− 196 °C), the cell survival significantly decreased.     

Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus

This study investigated the formation and utilization of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus. Time-course papaya juice fermentations were carried out using pure cultures of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 and a mixed culture of the two yeasts at a ratio of 1:1000 (R2:NCYC2251). Changes in S. cerevisiae cell population, Brix, sugar consumption and pH were similar in the mixed culture and in the S. cerevisiae monoculture. There was an early growth arrest of W. saturnus in the mixed culture fermentation. A range of volatile compounds were produced during fermentation including fatty acids, alcohols, aldehydes and esters and some volatile compounds including those initially present in the juice were utilized. The mixed culture fermentation of S. cerevisiae and W. saturnus benefited from the presence of both yeasts, with more esters being produced than the S. cerevisiae monoculture and more alcohols being formed than the W. saturnus monoculture. The study suggests that papaya juice fermentation with a mixed culture of S. cerevisiae and W. saturnus may be able to result in the formation of more complex aroma compounds and higher ethanol level than those using single yeasts.     

Influence of autochthonous Saccharomyces cerevisiae strains on volatile profile of Negroamaro wines

Saccharomyces cerevisiae is the yeast species predominating the alcoholic fermentation of grape must. The aim of this research was to evaluate the impact of indigenous S. cerevisiae strains biodiversity on the aroma of wines from Negroamaro grapes. Grapes collected in two different Negroamaro producing micro districts in Salento (Southern Italy), were subjected to natural fermentation and two indigenous S. cerevisiae populations were isolated. Fifteen strains for each of the two populations were selected and tested by micro fermentation assay in order to evaluate their specific contribute to the volatiles composition and sensory impact of the produced wines. The aromatic profile of wines obtained by each selected strain was characterized by different contents of acetates, ethyl esters of fatty acids, higher alcohols, thus showing to be related to the strains geographical origin. The sensorial analysis of wines produced by the six best performing strains confirmed that they are good candidates as industrial starter cultures, This study indicates that the use of a “microarea-specific” starter culture is a powerful tool to enhance the peculiarity of wines deriving from specific areas.     

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.     

Interactions between aroma compounds and whole mannoprotein isolated from Saccharomyces cerevisiae strains

The interactions of four wine aroma compounds (isoamyl acetate, hexanol, ethyl hexanoate, β-ionone) either with a whole mannoprotein extract or with mannoprotein fractions at a level encountered in wine (150 mg/L) were studied by dynamic and static headspace techniques. The mannoproteins were isolated from a synthetic medium subjected to an alcoholic fermentation with two enological yeast strains. They were fractionated by exclusion chromatography and characterized through glycosyl composition analysis.     

Inducing malolactic fermentation in Chardonnay musts and wines using different strains of Oenococcus oeni

Malolactic fermentations (MLF) were induced in a commercially prepared Washington State Chardonnay must to evaluate the influence of timing of inoculation and pre-culture conditions of Oenococcus oeni strains MCW, EQ-54, and WS-8. The must (pH 3.62, 21.5°Brix) was divided into lots and inoculated with Saccharomyces cerevisiae strain CY3079. Strains of O. oeni were pre-cultured by growing in diluted juice or by re-hydration of freeze-dried strains. Bacteria were inoculated into the musts before (Day 0) or after completion of the alcoholic fermentation (Day 22). Yeast populations exceeded 10⁷cfu/mL in all fermentations that proceeded to dryness. However, the viability of most strains of O. oeni quickly declined after inoculation regardless of the timing of inoculation or the strain used. MLF was induced in the wines inoculated with strains EQ-54 and WS-8 but not with MCW, and the rate depended on the time of inoculation. The method used to prepare bacterial starter cultures had no apparent influence on the completion of MLF. Values for volatile acidity were slightly higher (P< 0.05) in wines inoculated with O. oeni before alcoholic fermentation compared with those inoculated after alcoholic fermentation.     

Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae by UV-C light: Study of cell injury by flow cytometry

Flow cytometry (FCM) is a powerful tool for analyzing physiological characteristics of microorganisms on a single-cell basis and identifying heterogeneities within population. This work analyzed the UV-C induced damage on Escherichia coli ATCC 11229; Listeria innocua ATCC 33090 and Saccharomyces cerevisiae KE162 cells by applying flow cytometry technique. The UV-C doses, obtained by altering the exposure time and measured by the iodide-iodate chemical actinometer, ranged between 0 and 5 kJ/m². E. coli; L. innocua and S. cerevisiae populations were quantified by plate count technique. For flow cytometry studies, cells were labeled with fluorescein diacetate (FDA) for detecting membrane integrity and esterase activity, and with propidium iodide (PI) for monitoring membrane integrity. The results showed that mechanisms of cellular damage differed according to time of exposure to ultraviolet radiation and the organism tested. E. coli and S. cerevisiae sub-populations with PI increased within the first minutes of UV-C treatment, without much change afterwards. On the contrary, FCM was used to detect the inactivation of those L. innocua sub-populations of viable microorganisms (maintaining metabolic activity) which were non-culturable due to membrane rupture and thus not detectable by viable plate count technique.     

Genetic diversity of Oenoccoccus oeni isolated from wines treated with phenolic extracts as antimicrobial agents

Molecular techniques have been applied to study the evolution of wine-associated lactic acid bacteria from red wines produced in the absence and presence of antimicrobial phenolic extracts, eucalyptus leaves and almond skins, and to genetically characterize representative Oenococcus oeni strains. Monitoring microbial populations by PCR-DGGE targeting the rpoB gene revealed that O. oeni was, as expected, the species responsible for malolactic fermentation (MLF). Representative strains from both extract-treated and not-treated wines were isolated and all were identified as O. oeni species, by 16S rRNA sequencing. Typing of isolated O. oeni strains based on the mutation of the rpoB gene suggested a more favorable adaptation of L strains (n = 63) than H strains (n = 3) to MLF. Moreover, PFGE analysis of the isolated O. oeni strains revealed 27 different genetic profiles, which indicates a rich biodiversity of indigenous O. oeni species in the winery. Finally, a higher number of genetic markers were shown in the genome of strains from control wines than strains from wines elaborated with phenolic extracts. These results provide a basis for further investigation of the molecular and evolutionary mechanisms leading to the prevalence of O. oeni in wines treated with polyphenols as inhibitor compounds.     

Evaluation of different Saccharomyces cerevisiae strains for red winemaking. Influence on the anthocyanin,pyranoanthocyanin and non-anthocyanin phenolic content and colour characteristics of wines

The possible industrial use of three previously-selected Saccharomyces cerevisiae strains (1EV, 2EV and 7EV) has been studied in musts derived from Tempranillo and Cabernet Sauvignon. The anthocyanin, pyranoanthocyanin and non-anthocyanin phenolic content, and colour characteristics of the resulting wines have been compared to those of a commercial strain. Anthocyanins were the compounds most influenced by the yeast strain. Independently of the grape variety, wines derived from 2EV presented significantly higher anthocyanin concentrations than those derived from 1EV and 7EV, which presented similar contents. With the exception of hydroxycinnamic acids and derivatives, no particular influence of the yeast strain was observed on the remaining non-anthocyanin phenolic compounds (i.e, hydroxybenzoic acids and flavanols). Pyranoanthocyanins and metabolites resulting from the alcoholic fermentation such as tyrosol and tryptophol, seemed to be more influenced by the must composition and pH, and thus, by the grape variety, than by the yeast strain.