Montse Poblet

Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae.

The influence of fermentation temperature (from 15 to 35 degrees C) on a mixed strain population was studied. Mitochondrial DNA analysis was used to differentiate Saccharomyces cerevisiae strains and the frequency of each strain during the alcoholic fermentation was determined. The chemical analyses of resulting wines were carried out. The temperature determined how Saccharomyces strains developed and how effectively they fermented. Some strains performed better at high temperatures and others at low temperatures. The maximal population size was similar at all temperatures. At low temperatures, however, it was reached later though it remained constant throughout the alcoholic fermentation. On the other hand, viable cells decreased at high temperatures, especially at 35 degrees C. Obviously, the composition of the wines changed as the temperature of fermentation changed. At low temperatures, alcohol yield was higher. Secondary metabolites to alcoholic fermentation increased as the temperature increased. Glycerol levels were directly affected by temperature.

Yeast population dynamics in spontaneous fermentations: Comparison between two different wine-producing areas over a period of three years

Yeast ecology, biogeography and biodiversity are important and interesting topics of research. The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998). No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations. Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA. This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation. However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains.

Effects of fermentation temperature and Saccharomyces species on the cell fatty acid composition and presence of volatile compounds in wine

Low temperature alcoholic fermentations are becoming more frequent due to the wish to produce wines with more pronounced aromatic profiles. However, their biggest drawback is the high risk of stuck and sluggish fermentations. Changes in the plasma membrane composition may be an adaptive response to low temperature fermentations. The production of volatile compounds and the changes in the membrane fatty acids were determined by GC to show the degree of cell adaptation and performance at low temperatures (13 degrees C) taking 25 degrees C as reference. The tests were done in two strains of Saccharomyces cerevisiae and one strain of Saccharomyces bayanus. Low temperatures restricted yeast growth and lengthened the fermentations. The analysis of plasma membrane fatty acids showed that dry yeasts had similar levels of unsaturation, between 70% and 80%, with no medium-chain fatty acids (MCFA). Long-chain saturated fatty acids (SFA) were the most frequent membrane fatty acids throughout the fermentations. Lipid composition changed with the growth temperature. The optimal membrane fluidity at low temperatures was modulated by changes in the unsaturation degree in S. cerevisiae strains. In S. bayanus, however, this change in the unsaturated fatty acid (UFA) percentage was not observed at different growth temperatures but the concentration of MCFA at low fermentation temperatures was higher. Concentrations of volatile compounds were higher in wines produced at lower temperatures and depended on the strain.

Population dynamics of acetic acid bacteria during traditional wine vinegar production.

The population dynamics of acetic acid bacteria in traditional vinegar production was determined in two independent vinegar plants at both the species and strain level. The effect of barrels made of four different woods upon the population dynamics was also determined. Acetic acid bacteria were isolated on solid media and the species were identified by RFLP-PCR of 16S rRNA genes and confirmed by 16S rRNA gene sequencing, while strains were typed by ERIC-PCR and (GTG)(5)-rep-PCR. The most widely isolated species was Acetobacter pasteurianus, which accounted for 100% of all the isolates during most of the acetification. Gluconacetobacter europaeus only appeared at any notable level at the end of the process in oak barrels from one vinegar plant. The various A. pasteurianus strains showed a clear succession as the concentration of acetic acid increased. In both vinegar plants the relative dominance of different strains was modified as the concentrations of acetic acid increased, and strain diversity tended to reduce at the end of the process.

Effect of the nitrogen source on the fatty acid composition of Saccharomyces cerevisiae

The source and content of nitrogen in the medium are very important in the development of alcoholic fermentations since they both affect the growth of Saccharomyces cerevisiae. Furthermore, the composition of the growth medium and the environmental conditions are known to affect the cell membrane fatty acid composition. The aim of this work was to study how the nitrogen source affects the membrane fatty acid composition. A mixture of amino acids and ammonia delayed the yeast growth when a high content of yeast assimilable nitrogen was present in the media. Cells grown in the mixed nitrogen source had a lower content of total fatty acids with a higher unsaturation degree than cells grown on sole ammonia.

New insights into the capacity of commercial wine yeasts to grow on sparkling wine media. Factor screening for improving wine yeast selection.

During the production of sparkling wine, wine yeasts are subjected to many stress factors apart from ethanol, which lead to the need to achieve their acclimation in line with various industrial protocols. In the present work, 44 commercial wine Saccharomyces cerevisiae strains and one laboratory strain (BY4742) were firstly subjected to the influence of increasing concentrations of ethanol to cluster the yeasts using discriminant function analysis. Afterwards, non-inhibitory concentration (NIC) and minimum inhibitory concentration (MIC) were estimated, revealing some differences between 24 of these strains. Meanwhile, this study confirms the negative synergistic effect of low pH with ethanol on the maximum specific growth rate (μmax) and lag phase time. Moreover, a negative effect of increasing levels of glycerol in the growth medium was observed. Interestingly enough, an interactive positive effect was found between cysteine and medium-chain fatty acids (MCFA). While cysteine did not have a really significant effect in comparison to the control, it was able to restore the damage caused by MCFA, making the growth rate of cells recover and even reducing the formation of reactive oxygen species. Adequate culture aeration is also crucial for the composition of the cell fatty acid. The final results showed that few differences were observed between NIC and MIC estimations with respect to cells pre-cultured in the presence or absence of oxygen.

Effect of wood type and thickness on acetification kinetics in traditional vinegar production

Traditional vinegar production is a lengthy process which implies high operational risks and jeopardizes the organoleptic characteristics of the final product. In an effort to solve these problems without changing the traditional model, we modified the wood type and thickness of vinegar barrels. We acetified in triplicate in barrels made of acacia, cherry, chestnut, and oak and in three wood thicknesses (15, 20, and 25 mm) in two different vinegar plants. The operating volume was set at 60 L. Reducing wood thickness improved neither maximum acetification velocity or the total length of the process, and in some cases even worsened them. The process took longer in oak barrels than in other types of wood barrel in one of the vinegar plants. Therefore, the choice of wood is a parameter to be considered in the wine vinegar production.

New insights into the toxicity mechanism of octanoic and decanoic acids on Saccharomyces cerevisiae

Octanoic (C8) and decanoic (C10) acids are produced in hypoxic conditions by the yeast Saccharomyces cerevisiae as by‐products of its metabolism and are considered fermentation inhibitors in the presence of ethanol at acidic pH. This study aims to broaden our understanding of the physiological limits between toxicity and ester production in yeast cells. To this end, the non‐inhibitory concentration (NIC) and maximum inhibitory concentration (MIC) values were first established for C8 and C10 at physiological pH (5.8) without ethanol. The results showed that when these acids were added to culture medium at these values, they tended to accumulate in different cellular fractions of the yeast. While C8 was almost entirely located in the cell wall fraction, C10 was found in the endocellular fraction. Cell fatty acid detoxification was also different; while the esterification of fatty acids was more efficient in the case of C10, the peroxisome was activated regardless of which fatty acid was added. Furthermore, the study of the Pdr12 and Tpo1 transporters that evolved during the detoxification process revealed that C8 was mostly expelled by the Pdr12 carrier, which was related to higher β‐oxidative damage in the presence of endocellular C10. C10 is more toxic at lower concentrations than C8. Although they are produced by yeast, the resulting intracellular medium‐chain fatty acids (MCFAs) caused a level of toxicity which promoted cell death. However, MCFAs are involved in the production of beverage flavours. Copyright

Enhancing the tolerance of the Starmerella bacillaris wine strain to dehydration stress

A protocol was developed to obtain Starmerella bacillaris as an active dry wine yeast (ADWY), which will facilitate winemakers to realize sequential inoculations of grape must to improve wine complexity. In the present study, several compound solutions were analyzed during the dehydration–rehydration process for Starmerella bacillaris strains isolated from related environments of alcoholic beverages. The ADWY obtained from Starmerella bacillaris were evaluated in fermentations at laboratory scale, obtained by sequential- and co-inoculation with Saccharomyces cerevisiae; the fermentative and aromatic parameters were evaluated and discussed. Our results for one Starmerella bacillaris strain show that the enhancement of viability might lead to a 4-fold higher survival rate when cells are dried in the presence of 10% trehalose, followed by rehydration in 0.5% galactose solution. In co- and sequentially inoculated grape must fermentations with Saccharomyces cerevisiae, the laboratory-scale wines obtained with Starmerella bacillaris ADWY did not show major changes in terms of the main volatile compounds, but there was an improvement in the fermentation performance behavior. The present study paves the way to develop a protocol for developing Starmerella bacillaris as an ADWY.