Xylose and yeasts: A story beyond xylitol production.

Six different yeasts were used to study their metabolism of glucose and xylose, and mainly their capacity to produce ethanol and xylitol. The strains used were Candida guilliermondii, Debaryomyces hansenii, Saccharomyces cerevisiae, Kluyveromyces marxianus, Meyerozyma guilliermondii and Clavispora lusitaniae, four isolated from a rural mezcal fermentation facility. All of them produced ethanol when the substrate was glucose. When incubated in a medium containing xylose instead of glucose, only K. marxianus and M. guilliermondii were able to produce ethanol from xylose. On the other hand, all of them could produce some xylitol from xylose, but the most active in this regard were K. marxianus, M. guilliermondii, C. lusitaniae, and C. guilliermondii with the highest amount of xylitol produced. The capacity of all strains to take up glucose and xylose was also studied. Xylose, in different degrees, produced a redox imbalance in all yeasts. Respiration capacity was also studied with glucose or xylose, where C. guilliermondii, D. hansenii, K. marxianus and M. guilliermondii showed higher cyanide resistant respiration when grown in xylose. Neither xylose transport nor xylitol production were enhanced by an acidic environment (pH 4), which can be interpreted as the absence of a proton/sugar symporter mechanism for xylose transport, except for C. lusitaniae. The effects produced by xylose and their magnitude depend on the background of the studied yeast and the conditions in which these are studied.

Production of flavor compounds from rice bran by yeasts metabolisms of Kluyveromyces marxianus and Debaryomyces hansenii.

The aim of this study was to evaluate the biosynthesis of flavor compounds from rice bran by fermentation facilitated by Kluyveromyces marxianus and Debaryomyces hansenii. The growth of both yeasts was assessed by specific growth rates and doubling time. The biosynthesis of flavor compounds was evaluated by gas chromatography-olfactometry (GC-O), gas chromatography-mass spectrometry (GC-MS), and Spectrum™ sensory analysis. The specific growth rate (µ) and doubling time (td) of K. marxianus was calculated as 0.16/h and 4.21h, respectively, whereas that of D. hansenii was determined as 0.13/h and 5.33h, respectively. K. marxianus and D. hansenii produced significant levels of higher alcohols and acetate esters from rice bran. Results showed that K. marxianus can produce 827.27 µg/kg of isoamyl alcohol, 169.77 µg/kg of phenyl ethyl alcohol, and 216.08 µg/kg of phenyl ethyl acetate after 24-h batch fermentation. A significant amount of isovaleric acid was also synthesized by K. marxianus (4013 µg/kg) after the batch fermentation of 96 h. 415.64 µg/kg of isoamyl alcohol and 135.77 µg/kg of phenyl ethyl acetate was determined in rice bran fermented by D. hansenii after 24-h fermentation. Fermented cereals and rose were the characteristic flavor descriptors of the fermented rice bran samples. Rose flavor in fermented rice bran samples was found to be associated with phenyl ethyl alcohol, phenyl ethyl acetate, isoamyl acetate, and guaiacol. Thus, the findings of this study demonstrate that the valorization of rice bran can be achieved with the production of natural flavor compounds by yeast metabolism.

Non-conventional yeasts from fermented honey by-products: Focus on Hanseniaspora uvarum strains for craft beer production.

The increasing interest in novel beer productions focused on non-Saccharomyces yeasts in order to pursue their potential in generating groundbreaking sensory profiles. Traditional fermented beverages represent an important source of yeast strains which could express interesting features during brewing. A total of 404 yeasts were isolated from fermented honey by-products and identified as Saccharomyces cerevisiae, Wickerhamomyces anomalus, Zygosaccharomyces bailii, Zygosaccharomyces rouxii and Hanseniaspora uvarum. Five H. uvarum strains were screened for their brewing capability. Interestingly, Hanseniaspora uvarum strains showed growth in presence of ethanol and hop and a more rapid growth than the control strain S. cerevisiae US-05. Even though all strains showed a very low fermentation power, their concentrations ranged between 7 and 8 Log cycles during fermentation. The statistical analyses showed significant differences among the strains and underlined the ability of YGA2 and YGA34 to grow rapidly in presence of ethanol and hop. The strain YGA34 showed the best technological properties and was selected for beer production. Its presence in mixed- and sequential-culture fermentations with US-05 did not influence attenuation and ethanol concentration but had a significant impact on glycerol and acetic acid concentrations, with a higher sensory complexity and intensity, representing promising co-starters during craft beer production.

Use of Hanseniaspora guilliermondii and Hanseniaspora opuntiae to enhance the aromatic profile of beer in mixed-culture fermentation with Saccharomyces cerevisiae.

Beer production is predominantly carried out by Saccharomyces species, such as S. cerevisiae and S. pastorianus. However, the introduction of non-Saccharomyces yeasts in the brewing process is now seen as a promising strategy to improve and differentiate the organoleptic profile of beer. In this study, 17 non-Saccharomyces strains of 12 distinct species were isolated and submitted to a preliminary sensory evaluation to determine their potential for beer bioflavouring. Hanseniaspora guilliermondii IST315 and H. opuntiae IST408 aroma profiles presented the highest acceptability and were described as having 'fruity' and 'toffee' notes, respectively. Their presence in mixed-culture fermentations with S. cerevisiae US-05 did not influence attenuation and ethanol concentration of beer but had a significant impact in its volatile composition. Notably, while both strains reduced the total amount of ethyl esters, H. guilliermondii IST315 greatly increased the concentration of acetate esters, especially when sequentially inoculated, leading to an 8.2-fold increase in phenylethyl acetate ('rose', 'honey' aroma) in the final beverage. These findings highlight the importance of non-Saccharomyces yeasts in shaping the aroma profile of beer and suggest a role for Hanseniaspora spp. in improving it.

Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation.

Contamination with filamentous fungi during cocoa bean fermentation and drying reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop anti-fungal lactic acid bacteria (LAB)-yeast co-cultures by selecting anti-fungal strains best adapted to the cocoa bean fermentation process from 362 LAB and 384 yeast strains isolated from cocoa bean post-harvest processes. The applied multiphasic screening approach included anti-fungal activity tests in vitro and in vivo and assessment of the carbon metabolism and stress tolerance of the anti-fungal strains in a cocoa pulp simulation medium. The anti-fungal strains, Lactobacillus fermentum M017, Lb. fermentum 223, Hanseniaspora opuntiae H17, and Saccharomyces cerevisiae H290, were selected based on their high fungal growth inhibition capacity and their well-adapted metabolism. Up to seven filamentous fungal strains of the genera Aspergillus, Penicillium, and Gibberella were inhibited on average by 63 and 75% of the maximal inhibition zone by M017 and 223, respectively, and by 25 and 31% by the strains H17 and H290, respectively. Both Lb. fermentum strains converted the medium's glucose, fructose, and citric acid into 20.4-23.0 g/l of mannitol, 3.9-6.2 g/l acetic acid, and 8.6-10.3 g/l lactic acid, whereas the two yeast strains metabolized glucose and fructose to produce 7.4-18.4 g/l of ethanol. The Lb. fermentum strains were further characterized as particularly tolerant towards ethanol, acetic acid, and heat stress and both yeast strains tolerated high amounts of ethanol and lactic acid in the medium. Finally, the anti-fungal in vivo assays revealed that the two Lb. fermentum strains completely inhibited growth of the citrinin-producing strain, P. citrinum S005, and the potentially fumonisin-producing strain, G. moniliformis S003, on the surface of cocoa beans. Furthermore, growth of the aflatoxin-producer A. flavus S075 was inhibited after 10-14 days by all four selected anti-fungal strains, i.e. Lb. fermentum M017, Lb. fermentum 223, H. opuntiae H17, and Sacc. cerevisiae H290, at 51-95% when applied as single cultures and at 100% when the strains were combined into four co-cultures, each composed of a Lb. fermentum and one of the two yeast strains. As a conclusion, these four LAB-yeast co-cultures are recommended for future applications to limit the growth of filamentous fungi and the concomitant mycotoxin production during the fermentation of cocoa beans.

Optimization of fermentation-relevant factors: A strategy to reduce ethanol in red wine by sequential culture of native yeasts.

Current consumer preferences are determined by well-structured, full-bodied wines with a rich flavor and with reduced alcohol levels. One of the strategies for obtaining wines with reduced ethanol content is sequential inoculation of non-Saccharomyces and Saccharomyces cerevisiae yeasts. However, different factors affect the production of metabolites like ethanol, glycerol and acetic acid by inoculated yeasts. In order to obtain low alcohol wines without quality loss, the aims of our study were: i) to determine optimum conditions (fermentation temperature and time of permanence and initial inoculum size of the non-Saccharomyces population at the beginning of the process, prior to inoculation with S. cerevisiae); ii) to validate the optimized factors; and iii) to assess sensory quality of the wines obtained after validation. Two combinations of yeasts were used in this study: Hanseniaspora uvarum BHu9/S. cerevisiae BSc114 and Candida membranaefaciens BCm71/S. cerevisiae BSc114. Optimization of three fermentation factors that affect to non-Saccharomyces yeasts prior to S. cerevisiae inoculation was carried out using a Box-Behnken experimental design. Applying the models constructed by Response Surface Methodology, the lowest ethanol production by H. uvarum BHu9/S. cerevisiae BSc114 co-culture was obtained when H. uvarum BHu9 was inoculated 48 h 37 min prior to S. cerevisiae inoculation, at a fermentation temperature of 25 °C and at an initial inoculum size of 5 × 106 cells/mL. Lowest alcohol production with C. membranaefaciens BCm71/S. cerevisiae BSc114 was observed when C. membranaefaciens BCm71 was inoculated 24 h 15 min prior to S. cerevisiae at a fermentation temperature of 24.94 °C and at an initial inoculum size of 2.72 × 106 cells/mL. The optimized conditions of the two co-cultures were subsequently submitted to lab-scale validation. Both proposed strategies yielded ethanol levels that were significantly lower than control cultures (S. cerevisiae). Wines fermented with non-Saccharomyces/Saccharomyces co-cultures under optimized conditions were also associated with higher aromatic complexity characterized by the presence of red fruit aromas, whereas wines obtained with S. cerevisiae BSc114 were described by parameters linked with high ethanol levels.

Wine aroma response to different participation of selected Hanseniaspora uvarum in mixed fermentation with Saccharomyces cerevisiae.

Wine aroma response to a selected Hanseniaspora uvarum Yun268 strain was investigated using different inoculation strategies with commercial Saccharomyces cerevisiae yeast, namely, simultaneous fermentation (SiF), sequential fermentation (SeF), S. cerevisiae fermentation treated with extracellular extract of H. uvarum (EE), and pure S. cerevisiae fermentation (PF). Contributive volatiles in the perception of enhanced aroma traits were uncovered by partial least-squares regression. Results showed that controlled inoculation resulted into different amounts of H. uvarum Yun268, which distinctively affected the chemical and sensory profiles of wines. The concentration of aromatic compounds could be increased by H. uvarum Yun268 yeasts via high levels of ß-glucosidase activity and fatty acids. Terpenes, C13-norisoprenoids, acetate esters, ethyl esters, and fatty acids served as the impact volatiles that contributed to the enhanced aroma traits. SiF specifically increased the contents of C13-norisoprenoids, terpenes, and ethyl esters, while EE enhanced varietal volatile content rather than those of fermentative ones. However, excessive H. uvarum Yun268 in sequential inoculation elevated the concentrations of acetate esters and volatile phenols, triggering nail polish odor in Cabernet Sauvignon red wines.

Balsamic type varietal vinegar from cv. Xinomavro (Northen Greece). Optimization and scale-up of the alcoholic fermentation step using indigenous multistarters.

Taguchi design was used to examine the effect of parameters that should be optimized in order to control the alcoholic fermentation of the concentrated grape must (CGM) from cv. Xinomavro using the best-performing indigenous Hanseniaspora uvarum and Saccharomyces cerevisiae strains as multistarters. The "optimum" combination of conditions (cell ratio of H. uvarum/S. cerevisiae; inoculum size and inoculation time of S. cerevisiae; fermentation time and temperature) resulted in an alcoholic product that meets ethanol (79 g/kg) and residual sugar (164 g/kg) content requirements for further use in the production of balsamic type vinegar. Multistarter fermentation affected positively the varietal organoleptic traits of the fermented CGM. 5-(Hydroxymethyl)-furfural content emerged as a critical factor for the standardization of this process. Scaling up experiments in 12 L barrels verified findings from small scale in 100 mL flasks. The results of this work can be used as a prototype in further similar efforts.

Primary souring: A novel bacteria-free method for sour beer production.

In the beverage fermentation industry, especially at the craft or micro level, there is a movement to incorporate as many local ingredients as possible to both capture terroir and stimulate local economies. In the case of craft beer, this has traditionally only encompassed locally sourced barley, hops, and other agricultural adjuncts. The identification and use of novel yeasts in brewing lags behind. We sought to bridge this gap by bio-prospecting for wild yeasts, with a focus on the American Midwest. We isolated 284 different strains from 54 species of yeast and have begun to determine their fermentation characteristics. During this work, we found several isolates of five species that produce lactic acid and ethanol during wort fermentation: Hanseniaspora vineae, Lachancea fermentati, Lachancea thermotolerans, Schizosaccharomyces japonicus, and Wickerhamomyces anomalus. Tested representatives of these species yielded excellent attenuation, lactic acid production, and sensory characteristics, positioning them as viable alternatives to lactic acid bacteria (LAB) for the production of sour beers. Indeed, we suggest a new LAB-free paradigm for sour beer production that we term "primary souring" because the lactic acid production and resultant pH decrease occurs during primary fermentation, as opposed to kettle souring or souring via mixed culture fermentation.

Glycolytic Functions Are Conserved in the Genome of the Wine Yeast Hanseniaspora uvarum, and Pyruvate Kinase Limits Its Capacity for Alcoholic Fermentation.

Hanseniaspora uvarum (anamorph Kloeckera apiculata) is a predominant yeast on wine grapes and other fruits and has a strong influence on wine quality, even when Saccharomyces cerevisiae starter cultures are employed. In this work, we sequenced and annotated approximately 93% of the H. uvarum genome. Southern and synteny analyses were employed to construct a map of the seven chromosomes present in a type strain. Comparative determinations of specific enzyme activities within the fermentative pathway in H. uvarum and S. cerevisiae indicated that the reduced capacity of the former yeast for ethanol production is caused primarily by an ∼10-fold-lower activity of the key glycolytic enzyme pyruvate kinase. The heterologous expression of the encoding gene, H. uvarumPYK1 (HuPYK1), and two genes encoding the phosphofructokinase subunits, HuPFK1 and HuPFK2, in the respective deletion mutants of S. cerevisiae confirmed their functional homology.IMPORTANCEHanseniaspora uvarum is a predominant yeast species on grapes and other fruits. It contributes significantly to the production of desired as well as unfavorable aroma compounds and thus determines the quality of the final product, especially wine. Despite this obvious importance, knowledge on its genetics is scarce. As a basis for targeted metabolic modifications, here we provide the results of a genomic sequencing approach, including the annotation of 3,010 protein-encoding genes, e.g., those encoding the entire sugar fermentation pathway, key components of stress response signaling pathways, and enzymes catalyzing the production of aroma compounds. Comparative analyses suggest that the low fermentative capacity of H. uvarum compared to that of Saccharomyces cerevisiae can be attributed to low pyruvate kinase activity. The data reported here are expected to aid in establishing H. uvarum as a non-Saccharomyces yeast in starter cultures for wine and cider fermentations.

Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: a strategy to obtain wines with reduced ethanol content.

Ethanol content of wine has increased over the last decades as consequence of searching phenolic maturity, requiring increased grape maturity. This may result in the production of wines with excessive alcohol levels (sometimes more than 15% (v/v)), sluggish and stuck fermentations and excessive volatile acidity. Many strategies to reduce ethanol in wines are being studied, and microbial methods have some additional advantages. However, because of the broad intra- and interspecies variability, new selection criteria should be included. Therefore, the goal of the present work was to design and evaluate a simple and integral procedure for non-Saccharomyces yeast selection. This strategy allowed selection of yeasts that presented successful implantation in grape must with high alcohol potential and their use in co-cultures could reduce the ethanol in wines. A total of 114 native non-Saccharomyces yeasts were assayed to determine their respiratory, fermentative and physiological characteristics of enological interest. Hanseniaspora uvarum BHu9 and BHu11, H. osmophila BHo51, Starmerella bacillaris BSb55 and Candida membranaefaciens BCm71 were selected as candidates to design co-culture starters.

Influence of nitrogen sources on growth and fermentation performance of different wine yeast species during alcoholic fermentation.

In this study, the influence of twenty different single (i.e. 19 amino acids and ammonium sulphate) and two multiple nitrogen sources (N-sources) on growth and fermentation (i.e. glucose consumption and ethanol production) performance of Saccharomyces cerevisiae and of four wine-related non-Saccharomyces yeast species (Lachancea thermotolerans, Metschnikowia pulcherrima, Hanseniaspora uvarum and Torulaspora delbrueckii) was investigated during alcoholic fermentation. Briefly, the N-sources with beneficial effects on all performance parameters (or for the majority of them) for each yeast species were alanine, arginine, asparagine, aspartic acid, glutamine, isoleucine, ammonium sulphate, serine, valine and mixtures of 19 amino acids and of 19 amino acids plus ammonium sulphate (for S. cerevisiae), serine (for L. thermotolerans), alanine (for H. uvarum), alanine and asparagine (for M. pulcherrima), arginine, asparagine, glutamine, isoleucine and mixture of 19 amino acids (for T. delbrueckii). Furthermore, our results showed a clear positive effect of complex mixtures of N-sources on S. cerevisiae and on T. delbrueckii (although to a lesser extent) as to all performance parameters studied, whereas for L. thermotolerans, H. uvarum and M. pulcherrima, single amino acids affected growth and fermentation performance to the same extent as the mixtures. Moreover, we found groups of N-sources with similar effects on the growth and/or fermentation performance of two or more yeast species. Finally, the influences of N-sources observed for T. delbrueckii and H. uvarum resembled those of S. cerevisiae the most and the least, respectively. Overall, this work contributes to an improved understanding of how different N-sources affect growth, glucose consumption and ethanol production of wine-related yeast species under oxygen-limited conditions, which, in turn, may be used to, e.g. optimize growth and fermentation performance of the given yeast upon N-source supplementation during wine fermentations.

Interaction between Hanseniaspora uvarum and Saccharomyces cerevisiae during alcoholic fermentation.

During wine fermentation, Saccharomyces clearly dominate over non-Saccharomyces wine yeasts, and several factors could be related to this dominance. However, the main factor causing the reduction of cultivable non-Saccharomyces populations has not yet been fully established. In the present study, various single and mixed fermentations were performed to evaluate some of the factors likely responsible for the interaction between Saccharomyces cerevisiae and Hanseniaspora uvarum. Alcoholic fermentation was performed in compartmented experimental set ups with ratios of 1:1 and 1:9 and the cultivable population of both species was followed. The cultivable H. uvarum population decreased sharply at late stages when S. cerevisiae was present in the other compartment, similarly to alcoholic fermentations in non-compartmented vessels. Thus, cell-to-cell contact did not seem to be the main cause for the lack of cultivability of H. uvarum. Other compounds related to fermentation performance (such as sugar and ethanol) and/or certain metabolites secreted by S. cerevisiae could be related to the sharp decrease in H. uvarum cultivability. When these factors were analyzed, it was confirmed that metabolites from S. cerevisiae induced lack of cultivability in H. uvarum, however ethanol and other possible compounds did not seem to induce this effect but played some role during the process. This study contributes to a new understanding of the lack of cultivability of H. uvarum populations during the late stages of wine fermentation.

A yeast isolated from cashew apple juice and its ability to produce first- and second-generation ethanol.

The aim of this study was to isolate and identify an indigenous yeast from cashew apple juice (CAJ) and then use it in the production of first- and second-generation ethanol, using CAJ and the enzymatic hydrolysate of cashew apple bagasse (MCAB-OH), respectively. The isolated yeast was identified as belonging to the genus Hanseniaspora. Afterward, the effect of the medium initial pH on the production of ethanol from CAJ was evaluated in the range of 3.0 to 5.5, with its maximum ethanol production of 42 g L(-1) and Y P/S of 0.44 g g(-1) and 96 % efficiency. The effect of temperature (28-38 °C) on ethanol production was evaluated in a synthetic medium, and no difference in ethanol production in the temperature range evaluated (28-36 °C) was observed. At 32 °C, the yield, concentration, efficiency, and productivity of ethanol when using the CAJ medium were higher when compared to the results achieved for the synthetic medium. Regarding second-generation ethanol, the results showed that the yeast produced 24.37 g L(-1) of ethanol with an efficiency of 80.23 % and a productivity of 4.87 g L(-1) h(-1) at 5 h. Therefore, Hanseniaspora sp., isolated from CAJ, is a promising microorganism for the production of first- and second-generation ethanol.

Utilization of coffee by-products obtained from semi-washed process for production of value-added compounds.

The semi-dry processing of coffee generates significant amounts of coffee pulp and wastewater. This study evaluated the production of bioethanol and volatile compounds of eight yeast strains cultivated in a mixture of these residues. Hanseniaspora uvarum UFLA CAF76 showed the best fermentation performance; hence it was selected to evaluate different culture medium compositions and inoculum size. The best results were obtained with 12% w/v of coffee pulp, 1 g/L of yeast extract and 0.3 g/L of inoculum. Using these conditions, fermentation in 1 L of medium was carried out, achieving higher ethanol yield, productivity and efficiency with values of 0.48 g/g, 0.55 g/L h and 94.11% respectively. Twenty-one volatile compounds corresponding to higher alcohols, acetates, terpenes, aldehydes and volatile acids were identified by GC-FID. Such results indicate that coffee residues show an excellent potential as substrates for production of value-added compounds. H. uvarum demonstrated high fermentative capacity using these residues.

Diversity and physiological characterization of D-xylose-fermenting yeasts isolated from the Brazilian Amazonian Forest.

BACKGROUND: This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. METHODOLOGY/PRINCIPAL FINDINGS: A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L · h to 0.75 g/L · h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L · h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

Survival rate of wine-related yeasts during alcoholic fermentation assessed by direct live/dead staining combined with fluorescence in situ hybridization.

Real-time detection of microorganisms involved in complex microbial process, such as wine fermentations, and evaluation of their physiological state is crucial to predict whether or not those microbial species will be able to impact the final product. In the present work we used a direct live/dead staining (LDS) procedure combined with fluorescence in situ hybridization (FISH) to simultaneously assess the identity and viability of Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) during fermentations performed with single and mixed cultures. The population evolution of both yeasts was determined by plating and by LDS combined with species-specific FISH-probes labeled with Fluorescein. Since the FISH method involves the permeabilization of the cell membrane prior to hybridization and that it may influence the free diffusion of PI in and out of the cells, we optimized the concentration of this dye (0.5 µg of PI per 10(6) cells) for minimal diffusion (less than 2%). Fluorescent cells were enumerated by hemocytometry and flow cytometry. Results showed that the survival rate of Sc during mixed cultures was high throughout the entire process (60% of viable cells at the 9th day), while Hg began to die off at the 2nd day, exhibited 98% of dead cells at the 3rd day (45 g/l of ethanol) and became completely unculturable at the 4th day. However, under single culture fermentation the survival rate and culturability of Hg decreased at a much slower pace, exhibiting at the 7th day (67 g/l of ethanol) 8.7×10(4) CFU/ml and 85% of dead cells. Thus, our work demonstrated that the LDS-FISH method is able to simultaneously assess the viability and identity of these wine-related yeast species during alcoholic fermentation in a fast and reliable way. In order to validate PI-staining as a viability marker during alcoholic fermentation, we evaluated the effect of ethanol on the membrane permeability of Sc and Hg cells, as well as their capacity to recover membrane integrity after being exposed to different levels of ethanol (1%, 6%, 10%, 12% v/v). Results showed that while Sc cells were able to recover membrane integrity after ethanol exposure, Hg cells were not. However, under alcoholic fermentation Sc cells didn't recover membrane integrity after the mid-term (4-5 days) of alcoholic fermentation.

Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation.

Saccharomyces and non-Saccharomyces yeasts release enzymes that are able to transform neutral compounds of grape berries into active aromatic compounds, a process that enhances the sensory attributes of wines. So far, there exists only little information about enzymatic activity in mixed cultures of Saccharomyces and non-Saccharomyces during grape must fermentations. The aim of the present work was to determine the ability of yeasts to produce extracellular enzymes of enological relevance (ß-glucosidases, pectinases, proteases, amylases or xylanases) in pure and mixed Saccharomyces/non-Saccharomyces cultures during fermentation. Pure and mixed cultures of Saccharomyces cerevisiae BSc562, Hanseniaspora vinae BHv438 and Torulaspora delbrueckii BTd259 were assayed: 1% S. cerevisiae/99% H. vinae, 10% S. cerevisiae/90% H. vinae, 1% S. cerevisiae/99% T. delbrueckii and 10% S. cerevisiae/90% T. delbrueckii. Microvinifications were carried out with fresh must without pressing from Vitis vinifera L. c.v. Pedro Jiménez, an autochthonous variety from Argentina. Non-Saccharomyces species survived during 15-18days (BTd259) or until the end of the fermentation (BHv438) and influenced enzymatic profiles of mixed cultures. The results suggest that high concentrations of sugars did not affect enzymatic activity. ß-Glucosidase and pectinase activities seemed to be adversely affected by an increase in ethanol: activity diminished with increasing fermentation time. Throughout the fermentation, Saccharomyces and non-Saccharomyces isolates assayed produced a broad range of enzymes of enological interest that catalyze hydrolysis of polymers present in grape juice. Vinifications carried out by a pure or mixed culture of BTd259 (99% of T. delbrueckii) showed the highest production of all enzymes assayed except for ß-glucosidase. In mixed cultures, S. cerevisiae outgrew H. vinae, and T. delbrueckii was only detected until halfway the fermentation process. Nevertheless, their secreted enzymes could be detected throughout the fermentation process. Our results may contribute to a better understanding of the microbial interactions and the influence of some enzymes on vinification environments.

Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR.

Traditionally, it was assumed that non-Saccharomyces (NS) yeasts could only survive in the early stages of alcoholic fermentations. However, recent studies applying culture-independent methods have shown that NS populations persist throughout the fermentation process. The aim of the present work was to analyze and quantify Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) populations during alcoholic fermentations by plating and culture-independent methods, such as fluorescence in situ hybridization (FISH) and quantitative PCR (QPCR). Species-specific FISH probes labeled with fluorescein (FITC) were used to directly hybridize Sc and Hg cells from single and mixed cultures that were enumerated by epifluorescence microscopy and flow cytometry. Static and agitated fermentations were performed in synthetic grape juice and cell density as well as sugar consumption and ethanol production were determined throughout fermentations. Cell density values obtained by FISH and QPCR revealed the presence of high populations (107-108 cells/ml) of Sc and Hg throughout fermentations. Plate counts of both species did not show significant differences with culture-independent results in pure cultures. However, during mixed fermentations Hg lost its culturability after 4-6 days, while Sc remained culturable (about 108 cells/ml) throughout the entire fermentation (up to 10 days). The rRNA content of cells during mixed fermentations was also analyzed by flow cytometry in combination with FISH probes. The fluorescence intensity conferred by the species-specific FISH probes was considerably lower for Hg than for Sc. Moreover, the rRNA content of Hg cells, conversely to Sc cells, remained almost unchanged after boiling, which showed that rRNA stability is species-dependent.

Kinetic modelling of the sequential production of lactic acid and xylitol from vine trimming wastes.

A mathematical model describing the kinetics of the sequential production of lactic acid and xylitol from detoxified-concentrated vine trimming hemicellulosic hydrolysates by Lactobacillus rhamnosus and Debaryomyces hansenii, respectively, was developed from the basic principles of mass balance in two stages considering as main reactions: (1) glucose and xylose consumption by L. rhamnosus; and (2) xylitol and arabitol production by D. hansenii. The model allows to evaluate the yields and productivities under microaerobic and oxygen restricted conditions (in particular the effects caused by purging the oxygen with nitrogen), which were particularly important during the xylose to xylitol bioconversion by yeasts. The model was tested using experimental data obtained from detoxified-concentrated hemicellulosic hydrolysates, after CaCO3 addition in both types of fermentation processes, without purges (microaerobic conditions) or purging oxygen with nitrogen (oxygen-limited conditions) after sampling in order to reduce the oxygen dissolved. L. rhamnosus was removed by microfiltration before adding D. hansenii at the beginning of the second stage. Mass balance-based and logistic functions were successfully applied to develop the model of the system which properly predicts the consumption of sugars as well as the metabolites produced and yields. The dynamics of fermentation were also adequately described by the developed model.