Succinic acid production by wine yeasts and the influence of GABA and glutamic acid / Producción de ácido succínico por levaduras enológicas y la influencia del GABA y el ácido glutámico

As a consequence of alcoholic fermentation (AF) in wine, several compounds are released by yeasts, and some of them are linked to the general quality and mouthfeel perceptions in wine. However, others, such as succinic acid, act as inhibitors, mainly of malolactic fermentation. Succinic acid is produced by non-Saccharomyces and Saccharomyces yeasts during the initial stages of AF, and the presence of some amino acids such as γ-aminobutyric acid (GABA) and glutamic acid can increase the concentration of succinic acid. However, the influence of these amino acids on succinic acid production has been studied very little to date. In this work, we studied the production of succinic acid by different strains of non-Saccharomyces and Saccharomyces yeasts during AF in synthetic must, and the influence of the addition of GABA or glutamic acid or a combination of both. The results showed that succinic acid can be produced by non-Saccharomyces yeasts with values in the range of 0.2–0.4 g/L. Moreover, the addition of GABA or glutamic acid can increase the concentration of succinic acid produced by some strains to almost 100 mg/L more than the control, while other strains produce less. Consequently, higher succinic acid production by non-Saccharomyces yeast in coinoculated fermentations with S. cerevisiae strains could represent a risk of inhibiting Oenococcus oeni and therefore the MLF.(AU)

Succinic acid production by wine yeasts and the influence of GABA and glutamic acid.

As a consequence of alcoholic fermentation (AF) in wine, several compounds are released by yeasts, and some of them are linked to the general quality and mouthfeel perceptions in wine. However, others, such as succinic acid, act as inhibitors, mainly of malolactic fermentation. Succinic acid is produced by non-Saccharomyces and Saccharomyces yeasts during the initial stages of AF, and the presence of some amino acids such as γ-aminobutyric acid (GABA) and glutamic acid can increase the concentration of succinic acid. However, the influence of these amino acids on succinic acid production has been studied very little to date. In this work, we studied the production of succinic acid by different strains of non-Saccharomyces and Saccharomyces yeasts during AF in synthetic must, and the influence of the addition of GABA or glutamic acid or a combination of both. The results showed that succinic acid can be produced by non-Saccharomyces yeasts with values in the range of 0.2-0.4 g/L. Moreover, the addition of GABA or glutamic acid can increase the concentration of succinic acid produced by some strains to almost 100 mg/L more than the control, while other strains produce less. Consequently, higher succinic acid production by non-Saccharomyces yeast in coinoculated fermentations with S. cerevisiae strains could represent a risk of inhibiting Oenococcus oeni and therefore the MLF.

Microbial interactions in alcoholic beverages / Interacciones microbianas en bebidas alcohólicas

This review examines the different types of interactions between the microorganisms involved in the fermentation processes of alcoholic beverages produced all over the world from cereals or fruit juices. The alcoholic fermentation converting sugars into ethanol is usually carried out by yeasts, mainly Saccharomyces cerevisiae, which can grow directly using fruit sugars, such as those in grapes for wine or apples for cider, or on previously hydrolyzed starch of cereals, such as for beers. Some of these beverages, or the worts obtained from cereals, can be distilled to obtain spirits. Besides S. cerevisiae, all alcoholic beverages can contain other microorganisms and especially in spontaneous fermentation when starter cultures are not used. These other microbes are mostly lactic acid bacteria and other yeasts—the non-Saccharomyces yeasts. The interactions between all these microorganisms are very diverse and complex, as in any natural occurring ecosystem, including food fermentations. To describe them, we have followed a simplified ecological classification of the interactions. The negative ones are amensalism, by which a metabolic product of one species has a negative effect on others, and antagonism, by which one microbe competes directly with others. The positive interactions are commensalism, by which one species has benefits but no apparent effect on others, and synergism, by which there are benefits for all the microbes and also for the final product. The main interactions in alcoholic beverages are between S. cerevisiae and non-Saccharomyces and between yeasts and lactic acid bacteria. These interactions can be related to metabolites produced by fermentation such as ethanol, or to secondary metabolites such as proteinaceous toxins, or are feed-related, either by competition for nutrients or by benefit from released compounds during yeast autolysis...(AU)

Microbial interactions in alcoholic beverages.

This review examines the different types of interactions between the microorganisms involved in the fermentation processes of alcoholic beverages produced all over the world from cereals or fruit juices. The alcoholic fermentation converting sugars into ethanol is usually carried out by yeasts, mainly Saccharomyces cerevisiae, which can grow directly using fruit sugars, such as those in grapes for wine or apples for cider, or on previously hydrolyzed starch of cereals, such as for beers. Some of these beverages, or the worts obtained from cereals, can be distilled to obtain spirits. Besides S. cerevisiae, all alcoholic beverages can contain other microorganisms and especially in spontaneous fermentation when starter cultures are not used. These other microbes are mostly lactic acid bacteria and other yeasts-the non-Saccharomyces yeasts. The interactions between all these microorganisms are very diverse and complex, as in any natural occurring ecosystem, including food fermentations. To describe them, we have followed a simplified ecological classification of the interactions. The negative ones are amensalism, by which a metabolic product of one species has a negative effect on others, and antagonism, by which one microbe competes directly with others. The positive interactions are commensalism, by which one species has benefits but no apparent effect on others, and synergism, by which there are benefits for all the microbes and also for the final product. The main interactions in alcoholic beverages are between S. cerevisiae and non-Saccharomyces and between yeasts and lactic acid bacteria. These interactions can be related to metabolites produced by fermentation such as ethanol, or to secondary metabolites such as proteinaceous toxins, or are feed-related, either by competition for nutrients or by benefit from released compounds during yeast autolysis. The positive or negative effects of these interactions on the organoleptic qualities of the final product are also revised. Focusing mainly on the alcoholic beverages produced by spontaneous fermentations, this paper reviews the interactions between the different yeasts and lactic acid bacteria in wine, cider, beer, and in spirits such as tequila, mezcal and cachaça.

Yeasts Associated with Various Amazonian Native Fruits.

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.

Predominance of Lactobacillus plantarum Strains in Peruvian Amazonian Fruits.

The objective of this research was the identification and characterization of lactic acid bacteria (LAB) isolated from Peruvian Amazonian fruits. Thirty-seven isolates were obtained from diverse Amazonian fruits. Molecular characterization of the isolates was performed by ARDRA, 16S-23S ITS RFLP and rep-PCR using GTG5 primers. Identification was carried out by sequencing the 16S rDNA gene. Phenotypic characterization included nutritional, physiological and antimicrobial resistance tests. Molecular characterization by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and 16S-23S ITS RFLP resulted in four restriction profiles while GTG5 analysis showed 14 banding patterns. Based on the 16S rDNA gene sequence, the isolates were identified as Lactobacillus plantarum (75.7%), Weissella cibaria (13.5%), Lactobacillus brevis (8.1%), and Weissella confusa (2.7%). Phenotypic characterization showed that most of the isolates were homofermentative bacilli, able to ferment glucose, maltose, cellobiose, and fructose and grow in a broad range of temperatures and pH. The isolates were highly susceptible to ampicillin, amoxicillin, clindamycin, chloramphenicol, erythromicyn, penicillin, and tetracycline and showed great resistance to kanamycin, gentamycin, streptomycin, sulfamethoxazole/trimethoprim, and vancomycin. No proteolytic or amylolytic activity was detected. L. plantarum strains produce lactic acid in higher concentrations and Weissella strains produce exopolymers only from sucrose. Molecular methods allowed to accurately identify the LAB isolates from the Peruvian Amazonian fruits, while phenotypic methods provided information about their metabolism, physiology and other characteristics that may be useful in future biotechnological processes. Further research will focus especially on the study of L. plantarum strains.The objective of this research was the identification and characterization of lactic acid bacteria (LAB) isolated from Peruvian Amazonian fruits. Thirty-seven isolates were obtained from diverse Amazonian fruits. Molecular characterization of the isolates was performed by ARDRA, 16S-23S ITS RFLP and rep-PCR using GTG5 primers. Identification was carried out by sequencing the 16S rDNA gene. Phenotypic characterization included nutritional, physiological and antimicrobial resistance tests. Molecular characterization by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and 16S-23S ITS RFLP resulted in four restriction profiles while GTG5 analysis showed 14 banding patterns. Based on the 16S rDNA gene sequence, the isolates were identified as Lactobacillus plantarum (75.7%), Weissella cibaria (13.5%), Lactobacillus brevis (8.1%), and Weissella confusa (2.7%). Phenotypic characterization showed that most of the isolates were homofermentative bacilli, able to ferment glucose, maltose, cellobiose, and fructose and grow in a broad range of temperatures and pH. The isolates were highly susceptible to ampicillin, amoxicillin, clindamycin, chloramphenicol, erythromicyn, penicillin, and tetracycline and showed great resistance to kanamycin, gentamycin, streptomycin, sulfamethoxazole/trimethoprim, and vancomycin. No proteolytic or amylolytic activity was detected. L. plantarum strains produce lactic acid in higher concentrations and Weissella strains produce exopolymers only from sucrose. Molecular methods allowed to accurately identify the LAB isolates from the Peruvian Amazonian fruits, while phenotypic methods provided information about their metabolism, physiology and other characteristics that may be useful in future biotechnological processes. Further research will focus especially on the study of L. plantarum strains.

Sequential Inoculation of Native Non-Saccharomyces and Saccharomyces cerevisiae Strains for Wine Making.

The use of non-Saccharomyces yeast for wine making is becoming a common trend in many innovative wineries. The application is normally aimed at increasing aromas, glycerol, reducing acidity, and other improvements. This manuscript focuses on the reproduction of the native microbiota from the vineyard in the inoculum. Thus, native selected yeasts (Hanseniaspora uvarum, Metschnikowia pulcherrima, Torulaspora delbrueckii, Starmerella bacillaris species and three different strains of Saccharomyces cerevisiae) were inoculated sequentially, or only S. cerevisiae (three native strains together or one commercial) was used. Inoculations were performed both in laboratory conditions with synthetic must (400 mL) as well as in industrial conditions (2000 kg of grapes) in red winemaking in two different varieties, Grenache and Carignan. The results showed that all the inoculated S. cerevisiae strains were found at the end of the vinifications, and when non-Saccharomyces yeasts were inoculated, they were found in appreciable populations at mid-fermentation. The final wines produced could be clearly differentiated by sensory analysis and were of similar quality, in terms of sensory analysis panelists' appreciation.

Polysaccharides and Oligosaccharides Produced on Malvar Wines Elaborated with Torulaspora delbrueckii CLI 918 and Saccharomyces cerevisiae CLI 889 Native Yeasts from D.O. "Vinos de Madrid".

Polysaccharides and oligosaccharides released into Malvar white wines elaborated through pure, mixed, and sequential cultures with Torulaspora delbrueckii CLI 918 and Saccharomyces cerevisiae CLI 889 native yeasts from D.O. "Vinos de Madrid" were studied. Both fractions from different white wines were separated by high-resolution size-exclusion chromatography. Glycosyl composition and wine polysaccharide linkages were determined by GC-EI-MS chromatography. Molar-mass distributions were determined by SEC-MALLS, and intrinsic viscosity was determined by differential viscometer. Yeast species and type of inoculation have a significant impact on wine carbohydrate composition and structure. Mannose residues from mannoproteins were significantly predominant in those cultures where T. delbrueckii was present in the fermentation process in comparison with when pure cultures of S. cerevisiae were present in the fermenation process. Galactose residues from polysaccharides rich in arabinose and galactose presented greater values in pure cultures of S. cerevisiae, indicating that S. cerevisiae released fewer mannoproteins than T. delbrueckii. Moreover, we reported structural differences between mannoproteins released by T. delbrueckii CLI 918 and those released by S. cerevisiae CLI 889. These findings help to provide important information about the polysaccharides and oligosaccharides released from the cell walls of Malvar grapes and the carbohydrates released from each yeast species.

Yeast Monitoring of Wine Mixed or Sequential Fermentations Made by Native Strains from D.O. "Vinos de Madrid" Using Real-Time Quantitative PCR.

There is an increasing trend toward understanding the impact of non-Saccharomyces yeasts on the winemaking process. Although Saccharomyces cerevisiae is the predominant species at the end of fermentation, it has been recognized that the presence of non-Saccharomyces species during alcoholic fermentation can produce an improvement in the quality and complexity of the final wines. A previous work was developed for selecting the best combinations between S. cerevisiae and five non-Saccharomyces (Torulaspora delbrueckii, Schizosaccharomyces pombe, Candida stellata, Metschnikowia pulcherrima, and Lachancea thermotolorans) native yeast strains from D.O. "Vinos de Madrid" at the laboratory scale. The best inoculation strategies between S. cerevisiae and non-Saccharomyces strains were chosen to analyze, by real-time quantitative PCR (qPCR) combined with the use of specific primers, the dynamics of inoculated populations throughout the fermentation process at the pilot scale using the Malvar white grape variety. The efficiency of the qPCR system was verified independently of the samples matrix, founding the inoculated yeast species throughout alcoholic fermentation. Finally, we can validate the positive effect of selected co-cultures in the Malvar wine quality, highlighting the sequential cultures of T. delbrueckii CLI 918/S. cerevisiae CLI 889 and C. stellata CLI 920/S. cerevisiae CLI 889 and, mixed and sequential cultures of L. thermotolerans 9-6C combined with S. cerevisiae CLI 889.

Culture-dependent and independent techniques to monitor yeast species during cold soak carried out at different temperatures in winemaking.

Transformation of grape must into wine is a process that may vary according to the consumers' requirements. Application of cold soak prior to alcoholic fermentation is a common practice in cellars in order to enhance flavor complexity and extraction of phenolic compounds. However, the effect of this step on wine yeast microbiota is not well-known. The current study simultaneously analyzed the effect of different cold soak temperatures on the microbiological population throughout the process and the use of culture-dependent and independent techniques to study this yeast ecology. The temperatures assayed were those normally applied in wineries: 2.5, 8 and 12°C. PCR-DGGE allowed detection of the most representative species such as Hanseniaspora uvarum, Starmerella bacillaris and Saccharomyces cerevisiae. As could be expected, highest diversity indices were obtained at the beginning of each process, and survival of H. uvarum or S. bacillaris depended on the temperature. Our results are in agreement with those obtained with culture independent methods, but qPCR showed higher precision and a different behavior was observed for each yeast species and at each temperature assayed. Comparison of both culture-independent techniques can provide a general overview of the whole process, although DGGE does not reveal the diversity expected due to the reported problems with the sensitivity of this technique.

Yeast Biodiversity from DOQ Priorat Uninoculated Fermentations.

Climate, soil, and grape varieties are the primary characteristics of terroir and lead to the definition of various appellations of origin. However, the microbiota associated with grapes are also affected by these conditions and can leave a footprint in a wine that will be part of the characteristics of terroir. Thus, a description of the yeast microbiota within a vineyard is of interest not only to provide a better understanding of the winemaking process, but also to understand the source of microorganisms that maintain a microbial footprint in wine from the examined vineyard. In this study, two typical grape varieties, Grenache and Carignan, have been sampled from four different vineyards in the DOQ Priorat winegrowing region. Afterward, eight spontaneous alcoholic fermentations containing only grapes from one sampling point and of one variety were conducted at laboratory scale. The fermentation kinetics and yeast population dynamics within each fermentation experiment were evaluated. Yeast identification was performed by RFLP-PCR of the 5.8S-ITS region and by sequencing D1/D2 of the 26S rRNA gene of the isolates. The fermentation kinetics did not indicate clear differences between the two varieties of grapes or among vineyards. Approximately 1,400 isolates were identified, exhibiting high species richness in some fermentations. Of all the isolates studied, approximately 60% belong to the genus Hanseniaspora, 16% to Saccharomyces, and 11% to Candida. Other minor genera, such as Hansenula, Issatchenkia, Kluyveromyces, Saccharomycodes, and Zygosaccharomyces, were also found. The distribution of the identified yeast throughout the fermentation process was studied, and Saccharomyces cerevisiae was found to be present mainly at the end of the fermentation process, while Aureobasidium pullulans was isolated primarily during the first days of fermentation in three of the eight spontaneous fermentations. This work highlights the complexity and diversity of the vineyard ecosystem, which contains yeasts from different species. The description of this yeast diversity will lead to the selection of native microbiota that can be used to produce quality wines with the characteristics of the Priorat.

The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific.

The present study analyzes the lack of culturability of different non-Saccharomyces strains due to interaction with Saccharomyces cerevisiae during alcoholic fermentation. Interaction was followed in mixed fermentations with 1:1 inoculation of S. cerevisiae and ten non-Saccharomyces strains. Starmerella bacillaris, and Torulaspora delbrueckii indicated longer coexistence in mixed fermentations compared with Hanseniaspora uvarum and Metschnikowia pulcherrima. Strain differences in culturability and nutrient consumption (glucose, alanine, ammonium, arginine, or glutamine) were found within each species in mixed fermentation with S. cerevisiae. The interaction was further analyzed using cell-free supernatant from S. cerevisiae and synthetic media mimicking both single fermentations with S. cerevisiae and using mixed fermentations with the corresponding non-Saccharomyces species. Cell-free S. cerevisiae supernatants induced faster culturability loss than synthetic media corresponding to the same fermentation stage. This demonstrated that some metabolites produced by S. cerevisiae played the main role in the decreased culturability of the other non-Saccharomyces yeasts. However, changes in the concentrations of main metabolites had also an effect. Culturability differences were observed among species and strains in culture assays and thus showed distinct tolerance to S. cerevisiae metabolites and fermentation environment. Viability kit and recovery analyses on non-culturable cells verified the existence of viable but not-culturable status. These findings are discussed in the context of interaction between non-Saccharomyces and S. cerevisiae.

Fungal diversity in grape must and wine fermentation assessed by massive sequencing, quantitative PCR and DGGE.

The diversity of fungi in grape must and during wine fermentation was investigated in this study by culture-dependent and culture-independent techniques. Carignan and Grenache grapes were harvested from three vineyards in the Priorat region (Spain) in 2012, and nine samples were selected from the grape must after crushing and during wine fermentation. From culture-dependent techniques, 362 isolates were randomly selected and identified by 5.8S-ITS-RFLP and 26S-D1/D2 sequencing. Meanwhile, genomic DNA was extracted directly from the nine samples and analyzed by qPCR, DGGE and massive sequencing. The results indicated that grape must after crushing harbored a high species richness of fungi with Aspergillus tubingensis, Aureobasidium pullulans, or Starmerella bacillaris as the dominant species. As fermentation proceeded, the species richness decreased, and yeasts such as Hanseniaspora uvarum, Starmerella bacillaris and Saccharomyces cerevisiae successively occupied the must samples. The "terroir" characteristics of the fungus population are more related to the location of the vineyard than to grape variety. Sulfur dioxide treatment caused a low effect on yeast diversity by similarity analysis. Because of the existence of large population of fungi on grape berries, massive sequencing was more appropriate to understand the fungal community in grape must after crushing than the other techniques used in this study. Suitable target sequences and databases were necessary for accurate evaluation of the community and the identification of species by the 454 pyrosequencing of amplicons.

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.

Yeast population dynamics during prefermentative cold soak of Cabernet Sauvignon and Malbec wines.

Prefermentative cold soak is a widely used technique in red wine production, but the impact on the development of native yeast species is hardly described. The aim of this work was to analyse the dynamics and diversity of yeast populations during prefermentative cold soak in red wines. Three different temperatures (14 ± 1 °C; 8 ± 1 °C and 2.5 ± 1 °C) were used for prefermentative cold soak in Cabernet Sauvignon and Malbec grape musts. Saccharomyces and non-Saccharomyces populations during cold soak and alcoholic fermentation were analysed. In addition, the impact on chemical and sensory properties of the wines was examined. Yeast dynamics during prefermentative cold soak were temperature dependent. At 14 ± 1 °C, the total yeast population progressively increased throughout the cold soak period. Conversely, at 2.5 ± 1 °C, the yeast populations maintained stable during the same period. Prefermentative cold soak conducted at 14±1°C favoured development of Hanseniospora uvarum and Candida zemplinina, whereas cold soak conducted at 8 ± 1 °C favoured growth of Saccharomyces cerevisiae. At 2.5 ± 1 °C, no changes in yeast species were recorded. Acidity and bitterness, two sensory descriptors, appear to be related to wines produced with prefermentative cold soak carried out at 14 ± 1 °C. This fact could be associated with the increase in non-Saccharomyces during the prefermentation stage. Our results emphasise the importance of the temperature as a determinant factor to allow an increase in non-Saccharomyces population during prefermentative cold soak and consequently to modify sensorial attributes of wines as well as their sensorial impact.

Viable and culturable populations of Saccharomyces cerevisiae, Hanseniaspora uvarum and Starmerella bacillaris (synonym Candida zemplinina) during Barbera must fermentation.

The present study analyzed the viable and/or culturable populations of Saccharomyces cerevisiae, Hanseniaspora uvarum and Starmerella bacillaris (synonym Candida zemplinina) during laboratory grape must fermentation, in order to investigate the interaction between the three species considered. Firstly, population dynamics during wine fermentation were followed by culture-dependent techniques, and non-Saccharomyces yeast became non-culturable at late stages of fermentation when S. cerevisiae dominated. Four different culture-independent techniques were further applied to detect viable yeast cells at the late stage of fermentation. Both quantitative PCR techniques applied, namely ethidium monoazide bromide (EMA)-qPCR and Reverse Transcription (RT)-qPCR, detected H. uvarum and Starm. bacillaris at a concentration of 105 to 106cells/mL. These non-culturable cells had membranes impermeable to EMA and stable rRNA. The background signals from dead cells did not interfere with the quantification of viable cells in wine samples by EMA-qPCR technique. As a qualitative culture-independent technique, DGGE technique was coupled with EMA treatment (EMA-PCR-DGGE) or with RT (RT-PCR-DGGE). With EMA-PCR-DGGE non-Saccharomyces species during fermentation were detected although it was limited by the predominance of S. cerevisiae.

Monitoring of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris (synonym Candida zemplinina) populations during alcoholic fermentation by fluorescence in situ hybridization.

Various molecular approaches have been applied as culture-independent techniques to monitor wine fermentations over the last decade. Among them, those based on RNA detection have been widely used for yeast cell detection, assuming that RNA only exists in live cells. Fluorescence in situ hybridization (FISH) targeting intracellular rRNA is considered a promising technique for the investigation of wine ecology. For the present study, we applied the FISH technique in combination with epifluorescence microscopy and flow cytometry to directly quantify populations of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris during alcoholic fermentations. A new specific probe that hybridizes with eight species of Hanseniaspora genus and a second probe specific for Starm. bacillaris were designed, and the conditions for their application to pure cultures, mixed cultures, and wine samples were optimized. Single and mixed fermentations were performed with natural, concentrated must at two different temperatures, 15 °C and 25 °C. The population dynamics revealed that the Sacch. cerevisiae population increased to 10(7)-10(8)cells/ml during all fermentations, whereas H. uvarum and Starm. bacillaris tended to increase in single fermentations but remained at levels similar to their inoculations at 10(6)cells/ml in mixed fermentations. Temperature mainly affected the fermentation duration (slower at the lower temperature) but did not affect the population sizes of the different species. The use of these probes in natural wine fermentations has been validated.

Antimicrobial polyurethane thermosets based on undecylenic acid: synthesis and evaluation.

In the present study, plant oil-derived surface-modifiable polyurethane thermosets are presented. Polyol synthesis is carried out taking advantage of thiol-yne photopolymerization of undecylenic acid derivatives containing methyl ester or hydroxyl moieties. The prepared methyl ester-containing polyurethanes allow surface modification treatment to enhance their hydrophilicity and impart antimicrobial activity through the following two steps: i) grafting poly(propylene glycol) monoamine (Jeffamine M-600) via aminolysis and ii) Jeffamine M-600 layer complexation with iodine. The antimicrobial activity of the iodine-containing polyurethanes is demonstrated by its capacity to inhibit the growth of Staphylococcus aureus, and Candida albicans in agar media.

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.

Determination of viable wine yeast using DNA binding dyes and quantitative PCR.

The detection and quantification of wine yeast can be misleading due to under or overestimation of these microorganisms. Underestimation may be caused by variable growing rates of different microorganisms in culture media or the presence of viable but non-cultivable microorganisms. Overestimation may be caused by the lack of discrimination between live and dead microorganisms if quantitative PCR is used to quantify with DNA as the template. However, culture-independent methods that use dyes have been described to remove the DNA from dead cells and then quantify the live microorganisms. Two dyes have been studied in this paper: ethidium monoazide bromide (EMA) and propidium monoazide bromide (PMA). The technique was applied to grape must fermentation and ageing wines. Both dyes presented similar results on yeast monitoring. Membrane cell recovery was necessary when yeasts were originated from ethanol-containing media. When applied to grape must fermentation, differences of up to 1 log unit were seen between the QPCR estimation with or without the dye during the stationary phase. In ageing wines, good agreement was found between plating techniques and QPCR. Most of the viable cells were also culturable and no differences were observed with the methods, except for Zygosaccharomyces bailii and Dekkera bruxellensis where much higher counts were occasionally detected by QPCR. The presence of excess dead cells did not interfere with the quantification of live cells with either of the dyes.