Ana Rosa Gutiérrez

Ecology of spontaneous fermentation in one winery during 5 consecutive years

1999.The ecology of spontaneous fermentation in a new winery in La Rioja (Spain) was studied during 5 consecutive years using mitochondrial DNA restriction analysis. The number of different strains detected for each vintage and their appearance frequency varied from one year to another. A small number of strains were present in consecutive years, but the presence of each one varied in function of the specific year. Only one strain was present in all the 5 years studied. For the 1997 vintage, an unusual dominance of non‐Sacharomyces yeasts in the vigorous fermentation was detected; this may explain the abnormal analytical data for the wines of that year.

Presence of non-Saccharomyces yeasts in cellar equipment and grape juice during harvest time

The purpose of this study was to analyze the presence of different yeasts in the facilities of four wineries from the D.O.Ca. Rioja region in Spain. The study was conducted through the identification of the yeasts via the PCR-RFLP technique of the ITS region of rDNA. The diversity of non-Saccharomyces yeasts found in wineries has previously only been studied to a limited extent, despite the fact that these yeasts take part both in the start of spontaneous fermentation and in the changes which occur in the wines during their subsequent conservation. Most earlier studies carried out on cellar ecosystems have focussed on the clonal diversity of Saccharomyces cerevisiae. The results obtained in this study indicated that the presence of non-Saccharomyces yeasts in facilities is higher than that of the S. cerevisiae, with percentages of over 60% in all the wineries analyzed. Yeasts belonging to 10 genera and 18 species were isolated, but the only genera present in all four wineries were Cryptococcus, Pichia, and Saccharomyces. The Zygosaccharomyces bailii yeast responsible for taint was detected in one cleaned winery, in both the winemaking equipment and the fermenting must. It was also noted that the quantity and type of yeasts present in the facilities are related to the product used for cleaning them. It is also necessary to point out that the cleaning of the cellars prior to the reception of the grapes does not completely eliminate the yeasts present, so that these can subsequently become part of the vinification process.

The occurrence of fungi, yeasts and bacteria in the air of a Spanish winery during vintage

This research studies the presence of microorganisms of enological interest (yeasts, bacteria and molds) and their evolution in the air of a wine cellar. The samples were taken throughout the winemaking campaign (September-December) in a winery of the D.O.Ca. Rioja, Spain. They were collected using an airIDEAL atmosphere sampler from Biomerieux. For the isolation, specific selective media were used for each group of microorganisms. The results obtained indicate that the presence in the winery air of the various different microorganisms studied is directly related to the winemaking processes that are taking place in the winery. Thus, the number of molds present decreases once grapes have ceased to be brought into the winery. The maximum number of yeasts in the air is found when all the vats in the cellar are fermenting, while the lactic bacteria are not detected until the first malolactic fermentation begins. The species of yeasts and molds identified are also related to the winemaking processes. The coincidence of strains of Saccharomyces cerevisiae among those present in the vats during alcoholic fermentation and those isolated from the air, confirms the role of the latter as a transmitter of microorganisms.

Presence of lactic bacteria in the air of a winery during the vinification period

In this paper we have studied the presence and evolution in the winery air of the lactic bacteria responsible for malolactic fermentation. Sampling took place during the winemaking process (between September 2007 and July 2008) in a winery from the Rioja appellation in Spain. The results obtained indicated that the presence of these microorganisms in the atmosphere was detected when grapes were entering the winery, while malolactic fermentation was taking place, and when liquid containing bacteria was manipulated. The species and clones of the lactic bacteria identified were also related to those present in the vinification tanks at any given stage of the process.

Ecology of inoculated and spontaneous fermentations in Rioja (Spain) musts, examined by mitochondrial DNA restriction analysis

Analysis of mitochondrial DNA restriction patterns was used to study the introduction of a selected strain of Saccharomyces cerevisiae for fermentation of non-sterile musts of La Rioja (Spain). All of the isolates from the inoculated musts showed the restriction pattern of the selected strain. The same technique was used to study the spontaneous fermentation of musts, showing that a few strains were responsible for the fermentations. One of the strains identified from the spontaneous fermentations had been identified in a previous vintage.

Wine aromatic compound production and fermentative behaviour within different non-Saccharomyces species and clones.

Twenty‐five enological yeasts belonging to nine different species (Candida zeylanoides, Cryptococcus uzbekistanensis, Debaryomyces hansenii, Lachancea thermotolerans, Metschnikowia pulcherrima, Torulaspora delbrueckii, Williopsis pratensis, Zygosaccharomyces bailii and Saccharomyces cerevisiae) were screened for aroma formation and fermentative behaviour as part of a non‐Saccharomyces yeast selection programme.

Lactic acid bacteria communities in must, alcoholic and malolactic Tempranillo wine fermentations, by culture-dependent and culture-independent methods

The lactic acid bacteria (LAB) communities from must and through alcoholic (AF) and malolactic fermentations (MLF) of Tempranillo red wines were studied in ten wineries from the Designation of Origin Rioja during three consecutive vintages. A statistical study with data from both methods, PCR-DGGE and plating, was performed. Results showed that the LAB community in the D.O. Rioja was highly determined by the type of fermentation and also by the different stages within the winemaking, while other factors such as year, winery, or sampling subzones had not significant effect on the LAB species distribution. Three microbial families, seven genera, and 25 species were described in this research, and Lactobacillus was the most commonly detected genus before MLF. Curiously, genera and species not frequently detected in wines as Weissella, Fructobacillus, and Oenococcus kitaharae were identified during AF, and no-Oenococcus oeni species were described in some MLF by both methods. For the first time, two new O. oeni allelic groups were determined by 16S rDNA/DGGE being randomly adapted to the wine environment. Further studies targeted to understand the implication of the novel species, and O. oeni allelic groups in Rioja wine fermentations could be really interesting.

Screening of enzymatic activities within different enological non-Saccharomyces yeasts

Abstract Ninety-seven non-Saccharomyces wine yeast strains belonging to ten different genera and species (Candida spp. and Criptococcus spp.; Debaryomyces hansenii, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia kluyveri, Sporidiobolus salmonicolor, Torulaspora delbrueckii, Williopsis pratensis and Zygosaccharomyces bailii) were screened for 13 enzymes related to wine aroma, color and clarity. Understanding the yeasts’ influence in these wine characteristics provides a platform for selecting strains for their development as starter cultures and for the management of alcoholic fermentation. Most of the strains showed the presence of one or more enzymes of biotechnological interest. Our screening demonstrated several intraspecific differences within the yeast species investigated, indicating that strain selection is of great importance for their enological application, and also that some non-Saccharomyces that have not been thoroughly explored, may deserve further consideration. This research represents the first stage for selecting non-Saccharomyces strains to be used as a starter along with Saccharomyces cerevisiae to enhance some particular characteristics of wines.

Comparison of culture media for the recovery of airborne yeast in wineries

The direct air sampling impaction method on agar was evaluated using aerobiocollectors for the recovery of yeasts present in the winery air. Three culture media with different composition and specificity were studied. In addition, a resuscitation phase was included before the culture in the specificity medium [in the case of the Dekkera‐Brettanomyces Differential Medium (DBDM) medium]. Sampling was conducted at different times of the year and in different parts of the wineries, which were different in age and design. Both the Chloramphenicol Glucose Agar (CGA) and Agar Lysine AL media recovered yeasts from the air without any prior resuscitation phase. CGA was able to recover a higher number of colony‐forming units of yeasts than the other media. Consequently, to estimate the number of yeasts present in winery air, the best choice of medium would be CGA. The AL medium permitted the growth of the greatest range of genera and species. If the aim is to study the diversity of yeasts present in the air, the most suitable medium is AL. Neither CGA nor AL proved suitable for recovering yeasts of the Brettanomyces genus. The DBDM medium was the only one which provided sufficient specificity for their recovery and identification from the air, although their special characteristics made a prior protocol of resuscitation necessary.

Genetic and phenotypic intraspecific variability of non‐Saccharomyces yeasts populations from la rioja winegrowing region (Spain)

To determine the intraspecific genetic diversity within five non‐Saccharomyces yeast species and the diversity in phenotypic characteristic related to their technological properties.