Simultaneous Analysis of Organic Acids, Glycerol and Phenolic Acids in Wines Using Gas Chromatography-Mass Spectrometry.

Fermented beverages, particularly wines, exhibit variable concentrations of organic and phenolic acids, posing challenges in their accurate determination. Traditionally, enzymatic methods or chromatographic analyses, mainly high-performance liquid chromatography (HPLC), have been employed to quantify these compounds individually in the grape must or wine. However, chromatographic analyses face limitations due to the high sugar content in the grape must. Meanwhile, phenolic acids, found in higher quantities in red wines than in white wines, are typically analyzed using HPLC. This study presents a novel method for the quantification of organic acids (OAs), glycerol, and phenolic acids in grape musts and wines. The approach involves liquid-liquid extraction with ethyl acetate, followed by sample derivatization and analysis using gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) detection mode. The results indicated successful detection and quantification of all analyzed compounds without the need for sample dilution. However, our results showed that the method of adding external standards was more suitable for quantifying wine compounds, owing to the matrix effect. Furthermore, this method is promising for quantifying other metabolites present in wines, depending on their extractability with ethyl acetate. Fermented beverages, particularly wines, exhibit variable concentrations of organic and phenolic acids, posing challenges in their accurate determination. Traditionally, enzymatic methods or chromatographic analyses, mainly high-performance liquid chromatography (HPLC), have been employed to quantify these compounds individually in the grape must or wine. The approach of this proposed method involves (i) methoximation of wine compounds in a basic medium, (ii) acidification with HCl, (iii) liquid-liquid extraction with ethyl acetate, and (iv) silyl derivatization to analyze samples with gas chromatography-mass spectrometry (GC-MS) in ion monitoring detection mode (SIM). The results indicated successful detection and quantification of all analyzed compounds without the need for sample dilution. However, our results showed that the method of adding external standards was more suitable for quantifying wine compounds, owing to the matrix effect. Furthermore, this method is promising for quantifying other metabolites present in wines, depending on their extractability with ethyl acetate. In other words, the proposed method may be suitable for profiling (targeted) or fingerprinting (untargeted) strategies to quantify wine metabolites or to classify wines according to the type of winemaking process, grape, or fermentation.

Comparative study of inoculation strategies of Torulaspora delbrueckii and Saccharomyces cerevisiae on the performance of alcoholic and malolactic fermentations in an optimized synthetic grape must.

Progress in oenological biotechnology now makes it possible to control alcoholic (AF) and malolactic (MLF) fermentation processes for the production of wines. Key factors in controlling these processes and enhancing wine quality include the use of selected strains of non-Saccharomyces species, Saccharomyces cerevisiae, and Oenococcus oeni, as well as the method of inoculation (co-inoculation or sequential) and the timing of inoculation. In the present work, we investigated the effects of different inoculation strategies of two Torulaspora delbrueckii (Td-V and Td-P) strains followed by S. cerevisiae. Times (two, four, and six days) and types (co-inoculation and sequential) of inoculation were evaluated on the AF of a synthetic grape must. Furthermore, this synthetic medium was optimized by adding linoleic acid and ß-sitosterol to simulate the natural grape must and facilitate reproducible results in potential assays. Subsequently, the wines obtained were inoculated with two strains of Oenococcus oeni to carry out MLF. Parameters after AF were analysed to observe the impact of wine composition on the MLF performance. The results showed that the optimization of the must through the addition of linoleic acid and ß-sitosterol significantly enhanced MLF performance. This suggests that these lipids can positively impact the metabolism of O. oeni, leading to improved MLF efficiency. Furthermore, we observed that a 4-day contact period with T. delbrueckii leads to the most efficient MLF process and contributed to the modification of certain AF metabolites, such as the reduction of ethanol and acetic acid, as well as an increase in available nitrogen. The combination of Td-P with Oo-VP41 for 4 or 6 days during MLF showed that it could be the optimal option in terms of efficiency. By evaluating different T. delbrueckii inoculation strategies, optimizing the synthetic medium and studying the effects on wine composition, we aimed to gain insights into the relationship between AF conditions and subsequent MLF performance. Through this study, we aim to provide valuable insights for winemakers and researchers in the field of wine production and will contribute to a better understanding of the complex interactions between these species in the fermentation process.

Screening of Saccharomyces cerevisiae and Torulaspora delbrueckii strains in relation to their effect on malolactic fermentation.

The use of Torulaspora delbrueckii in the alcoholic fermentation (AF) of grape must is increasingly studied and used in the wine industry. In addition to the organoleptic improvement of wines, the synergy of this yeast species with the lactic acid bacterium Oenococcus oeni is an interesting field of study. In this work, 60 strain combinations were compared: 3 strains of Saccharomyces cerevisiae (Sc) and 4 strains of Torulaspora delbrueckii (Td) in sequential AF, and four strains of O. oeni (Oo) in malolactic fermentation (MLF). The objective was to describe the positive or negative relationships of these strains with the aim of finding the combination that ensures better MLF performance. In addition, a new synthetic grape must has been developed that allows the success of AF and subsequent MLF. Under these conditions, the Sc-K1 strain would be unsuitable for carrying out MLF unless there is prior inoculation with Td-Prelude, Td-Viniferm or Td-Zymaflore always with the Oo-VP41 combination. However, from all the trials performed, it appears that the combinations of sequential AF with Td-Prelude and Sc-QA23 or Sc-CLOS, followed by MLF with Oo-VP41, reflected a positive effect of T. delbrueckii compared to inoculation of Sc alone, such as a reduction in L-malic consumption time. In conclusion, the obtained results highlight the relevance of strain selection and yeast-LAB strain compatibility in wine fermentations. The study also reveals the positive effect on MLF of some T. delbrueckii strains.

Differentiation of Saccharomyces species by lipid and metabolome profiles from a single colony.

Yeast metabolism depends on growing conditions, which include the chemical composition of the medium, temperature and growth time. Historically, fatty acid profiles have been used to differentiate yeasts growing in liquid media. The present study determined the fatty acids of Saccharomyces species in colonies. Using the same method, the effect of that the number of colonies and growth time had on solid media allowed us to determine the metabolomic profiles of the cells. Our results showed that the lipid and metabolomic profiles of the cells evolved as the colony grew. Interestingly, some strains of Saccharomyces cerevisiae have been were differentiated using the fatty acid profile of a colony; concretely indeed EC1118 and QA23 strains were separated from ICV-K1 and BM4x4. The synthesis of saturated fatty acids was greater than that of unsaturated fatty acids during the first two days of cell growth on a solid medium compared to a liquid medium. Unsaturated fatty acids subsequently became predominant. Finally, this methodology could be useful for carrying out physiological studies in a complete or defined solid growth medium allowing the supplementation of compounds, which inhibit or activate the growth of yeasts.

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 ß-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.

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.

Metabolomic charactetization of yeast cells after dehydration stress

In this study, we analyzed the metabolite features of the yeasts Saccharomyces cerevisiae, Naumovia castellii, and Saccharomyces mikatae. The three species are closely related genetically but differ in their tolerance of desiccation stress. Specifically, we determined whether certain metabolites correlated with cell viability after stress imposition. The metabolomic profiles of these strains were compared before cell desiccation and after cell rehydration. In S. mikatae, the presence of lysine or glutamine during rehydration led to a 20% increase in survival whereas during dehydration the levels of both amino acids in this yeast were drastically reduced (AU)

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Metabolomic charactetization of yeast cells after dehydration stress.

In this study, we analyzed the metabolite features of the yeasts Saccharomyces cerevisiae, Naumovia castellii, and Saccharomyces mikatae. The three species are closely related genetically but differ in their tolerance of desiccation stress. Specifically, we determined whether certain metabolites correlated with cell viability after stress imposition. The metabolomics profiles of these strains were compared before cell desiccation and after cell rehydration. In S. mikatae, the presence of lysine or glutamine during rehydration led to a 20% increase in survival whereas during dehydration the levels of both amino acids in this yeast were drastically reduced.

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.

Application of molecular methods for routine identification of acetic acid bacteria.

Recently many new species of Acetic acid Bacteria have been described. The description and identification as new species was based on molecular techniques (sequencing of the 16S rRNA gene, DNA base ratio (% GC) determinations and DNA-DNA hybridisation) and phenotypic characterization. In the present paper, we propose a fast and reliable method for the identification most of the species currently described based on the RFLP-PCR of the 16S rRNA. According to the proposed protocol, 1 species can be identified with the use of a single enzyme, 13 with a combination of 2 enzymes, 2 species with a combination of 3 enzymes, 2 with a combination of 4 enzymes. To differentiate 5 more species RFLP-PCR of the ITS was also needed, after using 3 enzymes. Finally, a pair of species (Acetobacter pasteurianus and Acetobacter pomorum) could not be distinguished with the proposed method. However, doubts can be raised about their differentiation as separate species. Keeping these limitations in mind, the method is fast and reliable, allowing the processing of large number of samples in relatively short periods of time (less than 24 h after the isolation).

Application of molecular methods to demonstrate species and strain evolution of acetic acid bacteria population during wine production.

The growth of acetic acid bacteria on grapes or throughout the winemaking process influences the quality of wine, mainly because it increases the volatile acidity. The objective of this study was to analyse how the acetic acid bacteria population evolves in the changing environment of the grape surface and during wine fermentation. We have analysed the influence of yeast inoculation and SO2 addition on acetic acid bacteria populations. These bacteria were analysed at both the species and the strain level by molecular methods such as Restriction Fragment Length Polimorfism (RFLP) of amplified 16S rDNA, and amplification by polymerase chain reaction of Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) and Repetitive Extragenic Palindromic (REP-PCR). Our results show that the increases in population size are normally accompanied by a proliferation of Acetobacter aceti, which is the main species during fermentation. The diversity of strains is considerable in natural environments such as the grape surface. Changes in the environment during alcoholic fermentation substantially reduce the survival and the diversity of acetic acid bacteria. Few strains are able to survive these conditions and they seem to originate from both the grapes and the winery. To the best of our knowledge this is the first time that acetic acid bacteria are analysed at the strain level in grape surfaces and during winemaking.

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.

Effect of organic acids and nitrogen source on alcoholic fermentation: study of their buffering capacity.

The effect of tartaric acid and other organic acids on alcoholic fermentation was studied. Organic acids in media with high sugar concentrations and ammonium as the sole nitrogen source had an enormous impact on Saccharomyces cerevisiae metabolism during alcoholic fermentation. The main effect on yeast metabolism was the quick acidification of the media in the absence of organic acids. All of the organic acids used in this study (tartaric, malic, citric, and succinic acids) showed a buffering capacity, but not all of the acids had the same one. However, the results suggested that buffering should not be considered the only effect of organic acids on yeast metabolism. Nitrogen source also had a great influence on media pH. Ammonium consumption by yeasts produced a greater acidification of the media than when amino acids were used.

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.