Stages Of Wine Fermentation Research Articles

The Genus Metschnikowia in Enology.

Over the last decade, several non-Saccharomyces species have been used as an alternative yeast for producing wines with sensorial properties that are distinctive in comparison to those produced using only Saccharomyces cerevisiae as the classical inoculum. Among the non-Saccharomyces wine yeasts, Metschnikowia is one of the most investigated genera due to its widespread occurrence and its impact in winemaking, and it has been found in grapevine phyllospheres, fruit flies, grapes, and wine fermentations as being part of the resident microbiota of wineries and wine-making equipment. The versatility that allows some Metschnikowia species to be used for winemaking relies on an ability to grow in combination with other yeast species, such as S. cerevisiae, during the first stages of wine fermentation, thereby modulating the synthesis of secondary metabolites during fermentation in order to improve the sensory profile of the wine. Metschnikowia exerts a moderate fermentation power, some interesting enzymatic activities involving aromatic and color precursors, and potential antimicrobial activity against spoilage yeasts and fungi, resulting in this yeast being considered an interesting tool for use in the improvement of wine quality. The abovementioned properties have mostly been determined from studies on Metschnikowia pulcherrima wine strains. However, M. fructicola and M. viticola have also recently been studied for winemaking purposes.

Screening of Hanseniaspora Strains for the Production of Enzymes with Potential Interest for Winemaking

Some non-Saccharomyces yeasts, including Hanseniaspora, participate in the first stages of wine fermentation. Besides their importance in the wine production process some of these yeasts have been described to be potential producers of hydrolytic enzymes to industrial level. In this work, we pretend to evaluate the technological abilities of the Hanseniaspora strains deposited in the Spanish Type Culture Collection (CECT). First of all, we considered verification of the correct identification of the strains using several miniaturized biochemical systems and molecular techniques (PCR, RFLP and sequencing of the ribosomal D1/D2 region). The results allowed us to verify the correct adscription of the 26 strains included in this study, which exhibited concordant profiles of restriction with one of the three species described in previous studies (H. occidentalis, H. osmophila and H. valbyensis). Some other strains assigned to the species (H. uvarum, H. vineae and H. guilliermondii) showed at least two different profiles. The other objective of this study was to perform an initial screening to characterize both at quantitative and qualitative levels, the ability of these yeasts to produce valuable enzymes for wine fermentation (increase of aroma) and other applications. The more important enzymatic activities detected were β-glucosidase, β-xylosidase, and protease. The HU7, HU8, HV1, HV3, HO2 and HOC1 strains showed high levels of β-glucosidase and β-xylosidase activity, whereas some strains (HG1, HG3, HVA1, HOC 3 and HOC4 were useful for protease production.

An impaired ubiquitin ligase complex favors initial growth of auxotrophic yeast strains in synthetic grape must.

We used experimental evolution in order to identify genes involved in the adaptation of Saccharomyces cerevisiae to the early stages of alcoholic fermentation. Evolution experiments were run for about 200 generations, in continuous culture conditions emulating the initial stages of wine fermentation. We performed whole-genome sequencing of four adapted strains from three independent evolution experiments. Mutations identified in these strains pointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the preferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target proteins participating in different cellular processes, while Bul1p is an Rsp5p substrate adaptor specifically involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. While a loss-of-function mutation in BUL1 seems to be enough to confer a selective advantage under these assay conditions, this did not seem to be the case for RSP5 mutated strains, which required additional mutations, probably compensating for the detrimental effect of altered Rsp5p activity on essential cellular functions. The power of this experimental approach is illustrated by the identification of four independent mutants, each with a limited number of SNPs, affected within the same pathway. However, in order to obtain information relevant for a specific biotechnological process, caution must be taken in the choice of the background yeast genotype (as shown in this case for auxotrophies). In addition, the use of very stable continuous fermentation conditions might lead to the selection of a rather limited number of adaptive responses that would mask other possible targets for genetic improvement.

Effect of Fermentation Temperature and Culture Medium on Glycerol and Ethanol during Wine Fermentation

Yeast strain BH8, which exhibited significant differences from nine other yeast strains studied for glycerol production, was selected to investigate the effect of fermentation temperature (13 and 25°C) and culture medium (synthetic medium and grape must) on fermentation kinetics, yeast growth, and glycerol and ethanol synthesis at three different stages of wine fermentation. Yeast viability was better at 13°C than at 25°C in both growth media, and the more complex grape must enabled yeast cells to reach a higher population density. The accumulation of glycerol, glycerol-3-phosphate dehydrogenase (GPD) activity, and the level of expression of the GPD1 gene were highest in the initial stages of fermentation, which potentially counteracted the hyperosmotic stress caused by the high concentration of sugar in the media. More glycerol was produced during fermentation at 25°C than at 13°C and in grape must compared to the synthetic medium at both temperatures. Ethanol production was mainly affected by fermentation temperature. More ethanol was produced at 13°C than at 25°C, with lower expression of the ADH1 gene and level of ADH activity. The expression of the HSP104 and ALD6 genes was induced by ethanol stress in the final stages of fermentation. The amount of glycerol produced was not correlated to the production of acetic or succinic acid.

Comparing the transcriptomes of wine yeast strains: toward understanding the interaction between environment and transcriptome during fermentation

System-wide "omics" approaches have been widely applied to study a limited number of laboratory strains of Saccharomyces cerevisiae. More recently, industrial S. cerevisiae strains have become the target of such analyses, mainly to improve our understanding of biotechnologically relevant phenotypes that cannot be adequately studied in laboratory strains. Most of these studies have investigated single strains in a single medium. This experimental layout cannot differentiate between generally relevant molecular responses and strain- or media-specific features. Here we analyzed the transcriptomes of two phenotypically diverging wine yeast strains in two different fermentation media at three stages of wine fermentation. The data show that the intersection of transcriptome datasets from fermentations using either synthetic MS300 (simulated wine must) or real grape must (Colombard) can help to delineate relevant from "noisy" changes in gene expression in response to experimental factors such as fermentation stage and strain identity. The differences in the expression profiles of strains in the different environments also provide relevant insights into the transcriptional responses toward specific compositional features of the media. The data also suggest that MS300 is a representative environment for conducting research on wine fermentation and industrially relevant properties of wine yeast strains.

Indigenous wine killer yeasts and their application as a starter culture in wine fermentation

Wine yeast strains were isolated from seven fermentations of the red wines ‘Refošk’ and ‘Teran’, produced in the southwestern part of Slovenia. Among 613 isolated yeast strains, 22 expressed killer activity against the supersensitive strain Saccharomyces cerevisiae. Killer strains were isolated at different stages of wine fermentation but did not dominate in any of them. Species identification was based on the combination of RFLP analysis of an amplified rDNA region and biochemical–physiological tests. Killer isolates were identified as S. cerevisiae, Pichia anomala, Pichia kluyveri, Pichia pijperi, Hanseniaspora uvarum and Candida rugosa. Electrophoretic karyotyping was used to differentiate strains of the same species. Fermentation properties of four S. cerevisiae strains that possessed stable killer activity were characterized in fermentations of Malvasia must by studying their population dynamics and chemical composition and by sensory analysis of the wines produced. In order to compare the results, spontaneous fermentations and fermentations induced by commercial yeast starters were performed concomitantly. The local killer strain SS12/10 showed the best fermentation properties and produced wine with favourable characteristics.