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.

Impact of rare yeasts in Saccharomyces cerevisiae wine fermentation performance: Population prevalence and growth phenotype of Cyberlindnera fabianii, Kazachstania unispora, and Naganishia globosa.

Saccharomyces cerevisiae is a highly fermentative species able to complete the wine fermentation. However, the interaction with other non-Saccharomyces yeasts can determine the fermentation performance of S. cerevisiae. We have characterised three rare non-Saccharomyces yeasts (Cyberlindnera fabianii, Kazachstania unispora and Naganishia globosa), studying their impact on S. cerevisiae fitness and wine fermentation performance. Using a wide meta-taxonomic dataset of wine samples, analysed through ITS amplicon sequencing, we show that about a 65.07% of wine samples contains Naganishia spp., a 27.21% contains Kazachstania spp., and only a 4.41% contains Cyberlindnera spp; in all cases with average relative abundances lower than 1% of total fungal populations. Although the studied N. globosa strain showed a limited growth capacity in wine, both K. unispora and C. fabianii showed a similar growth phenotype to that of S. cerevisiae in different fermentation conditions, highlighting the outstanding growth rate values of K. unispora. In mixed fermentations with S. cerevisiae, the three yeast species affected co-culture growth parameters and wine chemical profile (volatile compounds, polysaccharides and proteins). K. unispora DN201 strain presents an outstanding capacity to compete with S. cerevisiae strains during the first stage of wine fermentation, causing stuck fermentations in both synthetic and natural grape musts.

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.