Study of the authenticity of commercial wine yeast strains by molecular techniques

Fernández-Espinar, M. Teresa; López-Alonso, Victoria; Ramón, Daniel; Bartra, Enric; Querol, Amparo

doi:10.1016/s0168-1605(01)00502-5

Cited by 102 publications

(80 citation statements)

References 33 publications

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“…However, S. pastorianus is only present in beer making and S. paradoxus has been isolated only once in wine (25). S. cerevisiae and S. bayanus are by far the most common species in wine fermentation, but in a previous work, we showed that all of the commercial S. bayanus strains characterized (12 strains) were in fact S. cerevisiae as result of incorrect classifications (12). This is why we have considered interesting design primers for the specific identification of strains belonging the species S. cerevisiae.…”

Section: Resultsmentioning

confidence: 88%

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Martorell

Querol

Fernández-Espinar

2005

Appl Environ Microbiol

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Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 ؋ 10 5 to 3.8 CFU/ml in sweet wine and from 5 ؋ 10 6 to 50 CFU/ml in red wine.Wine can become a growth substrate for a range of undesirable yeast species, both during and after fermentation. Uncontrolled yeast growth at either of these two stages can alter the chemical composition of wine, detracting from its sensory properties of appearance, aroma, and flavor. If these faults are severe, the wine is rejected by consumers. Thus, wine spoilage constitutes an important concern to wine producers.It is well known that Saccharomyces cerevisiae plays a beneficial role in wine fermentation, in which it is the predominant species. Nevertheless, S. cerevisiae is able to spoil wine after fermentation if the yeast is not properly eliminated or if the bottle is contaminated by yeast cells present in the wine bottling line or in the cork. S. cerevisiae is an important spoilage yeast because it resists high ethanol concentrations. It has been found mainly in sweet wines, where fermentable sugar can support growth, and also in semidry bottled wines.It is important to detect spoilage yeasts quickly to enable wineries to intervene rapidly and effectively. Methods based on PCR appear to be the best alternative. In the case of S. cerevisiae, several methods have been shown suitable for the detection of this species. This is the case of the restriction analysis of the rRNA region spanning the 5.8S gene and the two internal transcribed spacers (ITSs) (5.8S-ITS region). The amplification patterns of S. cerevisiae when its DNA is digested with the endonuclease CfoI, HaeIII, or HinfI identify this species accurately. These data are summarized in references 10 and 13 and are available online (http://www.yeast-id.com). Masneuf et al. (18) developed a similar system based on PCR amplification and subsequent restriction analysis using the nuclear gene MET2. These author...

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Section: Resultsmentioning

confidence: 88%

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Martorell

Querol

Fernández-Espinar

2005

Appl Environ Microbiol

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“…Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) is faster and easier than other methods (23,31). Digestion of mtDNA with restriction enzymes such as HinfI or RsaI is associated with a high rate of polymorphism and has been used to study the authenticity of commercial wine yeast strains (14,40,41). A PCR method based on variations found on the mitochondrial COX1 (a gene coding for cytochrome oxidase) intron number and position has been validated to distinguish S. cerevisiae strains allowing researchers to monitor the evolution of wine fermentations conducted by commercial active dry yeast (27).…”

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confidence: 99%

Biochemical and Molecular Characterization ofSaccharomyces cerevisiaeStrains Obtained from Sugar-Cane Juice Fermentations and Their Impact in Cachaça Production

Oliveira

Vicente

Fietto

et al. 2008

Appl Environ Microbiol

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Saccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H 2 S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5,5؆-trifluor-D,L-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard.Cachaça (pronounced "kha-sha-ssa"), the sugar cane spirit, is the most popular distilled beverage produced in Brazil. The annual production reaches 1.3 billion liters, with 15% being produced in more than 8,500 distilleries in the state of Minas Gerais.Traditional cachaça production relies on a spontaneous fermentative process that is mediated by the microbiota present in the cane juice wort and on the surface of equipments used in the productive process. It has been already demonstrated that in such systems there occurs a succession of yeasts, with Saccharomyces cerevisiae being the predominant species. Cachaça quality depends on the ecology of the microbial populations during an initial spontaneous fermentation (18,29,31,32,39). The fermentative process occurs through a continuously open fermentative process which is completed within 24 h and generally takes place from May to November, corresponding to the sugar cane harvesting period.Considering the conditions of production usually found in the cachaça distilleries, fermenting yeast populations have to face different types of stress (osmotic, high temperature, and high ethanol concentration). Besides, they might also present some characteristics such as a good fermentative power, no H 2 S production, killer activity, flocculation ability, and production of flavoring compounds. Taking all of these factors into account, we have developed a strategy to select yeast strains with appropriated characteristics to produce cachaça with potentially higher-quality standards (52). Parallel to the selection and development of S. cerevisiae strains toward ethanolic fermentations, molecular methods were developed and validated to study...

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“…Different methods are available to differentiate most commercial wine yeast strains, e.g. RFLP analysis of mt DNA (Fernandez-Espinar et al, 2001), PCR profile analysis of δ sequences (Lavallée et al, 1994;Legras and Karst, 2003) and fingerprinting of microsatellites (Pérez et al, 2001). As mentioned in the Introduction, these methods generate band patterns resulting from strain polymorphisms sometimes difficult to compare and reproduce, especially in the case of the PCR-based methods.…”

Section: Discussionmentioning

confidence: 99%

23S RNA‐derived replicon as a ‘molecular tag’ for monitoring inoculated wine yeast strains

Esteban

Rodríguez-Cousiño

2008

Yeast

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In this study we have developed a useful method to identify a particular yeast strain within a mixture of strains during must fermentation, based on the presence or absence of a stable genetic element derived from Saccharomyces cerevisiae 23S RNA autonomous replicon. 23S RNA is a natural virus-like RNA replicon present in some S. cerevisiae strains, which encodes only its own RNA-dependent RNA polymerase named p104. A modified version of 23S RNA (23S-tagged RNA) was generated after transformation of S. cerevisiae wine strains with a launching plasmid, where six nucleotides were changed in the 23S RNA cDNA sequence without modifying the amino acid sequence of p104 RNA polymerase. Once generated, the 23S-tagged RNA can replicate autonomously (without the plasmid), is very stable, is present in high copy number in stationary phase or nitrogen-starved cells and confers no phenotype to the host, like the endogenous 23S RNA replicon. However, it can be distinguished from endogenous 23S RNA by reverse transcription followed by polymerase chain reaction (RT-PCR) with specific oligonucleotide primers. 23S RNA-derived replicon can be used to tag wine yeast strains in order to monitor easily their prevalence over endogenous strains during wine fermentation.

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Study of the authenticity of commercial wine yeast strains by molecular techniques

Cited by 102 publications

References 33 publications

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Biochemical and Molecular Characterization ofSaccharomyces cerevisiaeStrains Obtained from Sugar-Cane Juice Fermentations and Their Impact in Cachaça Production

23S RNA‐derived replicon as a ‘molecular tag’ for monitoring inoculated wine yeast strains

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