V. Loureiro

Spoilage yeasts in the wine industry.

Yeasts play a central role in the spoilage of foods and beverages, mainly those with high acidity and reduced water activity (a(w)). A few species are capable of spoiling foods produced according to good manufacturing practices (GMPs). These can survive and grow under stress conditions where other microorganisms are not competitive. However, many of the aspects determining yeast spoilage have yet to be clarified. This critical review uses the wine industry as a case study where serious microbiological problems are caused by yeasts. First, the limitations of the available tools to assess the presence of spoilage yeasts in foods are discussed. Next, yeasts and factors promoting their colonisation in grapes and wines are discussed from the ecological perspective, demonstrating that a deeper knowledge of vineyard and winery ecosystems is essential to establish the origin of wine spoilage yeasts, their routes of contamination, critical points of yeast infection, and of course, their control. Further, zymological indicators are discussed as important tools to assess the microbiological quality of wines, although they are rarely used by the wine industry. The concepts of the susceptibility of wine to spoilage yeasts and wine stability are addressed based on scientific knowledge and industrial practices for monitoring yeast contamination. A discussion on acceptable levels of yeasts and microbiological criteria in the wine industry is supported by data obtained from wineries, wholesalers, and the scientific literature.Finally, future directions for applied research are proposed, involving collaboration between scientists and industry to improve the quality of wine and methods for monitoring the presence of yeast.

The wine proteins

Proteins are typically present in wines in low concentrations, contributing little to their nutritive value. However, they assume a considerable technological and economical importance because they greatly affect the clarity and stability of wines. Although exhibiting a large diversity, the majority of the wine proteins are structurally related and have been identified as pathogenesis related (PR) proteins. Thus, different wines are essentially composed by identical sets of polypeptides. They derive from the grape pulp, and survive the vinification process simply because they are highly resistant to proteolysis and to the low pH characteristic of wines. There is increasing evidence suggesting that although protein-dependent, the development of turbidity in wines is controlled by a number of factors of non-protein origin, such as polyphenols, the wine pH and the presence of polysaccharides. A variety of procedures has been developed and tested for the specific removal of proteins from wines. Even though bentonite fining is nonspecific and can impair the quality of wine, it remains the only effective method to stabilize wines.

Development and use of a new medium to detect yeasts of the genera Dekkera/Brettanomyces

Aims: The objectives of this work were to develop a selective and/or differential medium able to efficiently recover Dekkera/Brettanomyces sp. from wine‐related environments and to determine the relationship between these yeasts and the 4‐ethylphenol content in a wide range of wines.

Identification of yeasts isolated from wine-related environments and capable of producing 4-ethylphenol

The ability to produce 4-ethylphenol from the substrate p-coumaric acid in synthetic media was evaluated for several yeast species associated with wine production. Molar conversion rates as high as 90% were found by only Dekkera bruxellensis, D. anomala and by some unidentified strains isolated from wine-related environments. Other unidentified strains produced traces of 4-ethylphenol. All unidentified strains showed the same cultural characteristics as D. bruxellensis when grown on DBDM (Dekkera/Brettanomyces differential medium) agar. The determination of long-chain fatty acid compositions and the utilization of peptide nucleic acid (PNA) probes specific for D. bruxellensis showed that the unidentified strains did not belong to this species. Further identification, by restriction pattern generated from PCR-amplification of the 5.8S rRNA gene and the two internal transcribed spacers (ITS), assigned the unidentified strains to Candida cantarelli, C. wickerhamii, Debaryomyces hansenii, Kluyveromyces lactis and Pichia guilliermondii. However, only some strains of P. guilliermondii were capable of converting p-coumaric acid into 4-ethylphenol with efficiencies close to those observed in D. bruxellensis and D. anomala.

Spoilage yeasts in foods and beverages: characterisation and ecology for improved diagnosis and control

Foods and drinks with high solute concentration (fruit juices and concentrates, marzipan, salted and dry-cured meats, olives and cheeses), and foods containing preservatives (wine, beverages and sauces) were selected in order to characterise the contaminant yeast flora by rapid typing techniques. These included the testing of several types of molecular methods (e.g. RFLP, DNA-fingerprinting, PCR-based techniques), analysis of long-chain fatty acids and of isoenzymes. The PCR-based detection methods enabled a faster detection of emerging specific spoilers at earlier stages of processing. Fatty acid characterisation allowed the assessment of the most frequent types of contamination yeasts and supplied the information for the definition of relevant zymological indicators. A selected group of strains was used for further studies of mechanisms underlying the resistance/tolerance of yeasts towards preservatives (weak acids) and other stress factors (temperature, high sugar and salt concentrations). This study enabled the acquisition of data on the basic biology of yeasts used in the development of differential and selective media for Zygosaccharomyces bailii, Yarrowia lipolytica, Kluyveromyces marxianus and Dekkera sp.

Evaluation of the inhibitory effect of dimethyl dicarbonate (DMDC) against wine microorganisms

Several microbial species associated with wine were challenged against increasing concentrations of dimethyl dicarbonate (DMDC). The concentration inducing complete cell death upon addition to red wine was regarded as the minimum inhibitory concentration (MIC). In dry red wines with 12% (v/v) ethanol and pH 3.50, the inactivation depended on the initial cell concentration. For an initial inoculum of 500 CFU/ml, the MIC of the yeasts species Schizosaccharomyces pombe, Dekkera bruxellensis, Saccharomyces cerevisiae and Pichia guilliermondii was 100mg/l. The most sensitive strains belong to Zygosaccharomyces bailii, Zygoascus hellenicus and Lachancea thermotolerans, with MIC of 25mg/l DMDC. For inoculation rates of about 10(6)CFU/ml, the maximum dose of DMDC legally authorized (200mg/l) was not effective against the most resistant species. The addition of 100mg/l potassium metabisulphite (PMB), equivalent to 1mg/l molecular sulphur dioxide, increased the inactivation effect of 100mg/l DMDC over initial yeast populations of 10(6)CFU/ml but did not fully kill S. pombe and S. cerevisiae. Lactic acid and acetic acid bacteria were not killed by the addition of 300 mg/l of DMDC. Trials performed in wines before bottling showed that in most samples indigenous bacterial populations were not affected by 200mg/l DMDC. Therefore, under winery practice, DMDC at the maximum dose legally permitted may be regarded as an efficient preservative to control low contamination rates of yeasts but ineffective against lactic acid and acetic acid bacteria.

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

ABSTRACT Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid,p-hydroxyphenylethanol, andp-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol andp-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol andp-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde,p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation ofp-hydroxyphenylacetaldehyde, which is oxidized top-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn2+accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn2+. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.

Sour rot‐damaged grapes are sources of wine spoilage yeasts

Yeast species of sound and sour rot-damaged grapes were analysed during fermentation and grape ripening in the vineyard, using general and selective culture media. During 2003 and 2004 vintages, microvinifications were carried out with sound grapes to which different amounts of grapes with sour rot were added. The wine spoilage species Zygosaccharomyces bailii was only recovered during fermentations with sour rot, reaching 5.00 log CFU mL(-1) (2003) and 2.48 log CFU mL(-1) (2004) at the end of fermentation. The study of yeast populations during the sour rot ripening process (2005 vintage) showed that the veraison-damaged grapes always exhibited higher total yeast counts and a much greater diversity of species. From a total of 22 ascomycetous species, 17 were present only in damaged grapes. The most frequent species were Issatchenkia occidentalis and Zygoascus hellenicus. The spoilage species Z. bailii and Zygosaccharomyces bisporus were consistently isolated exclusively from damaged grapes. This work demonstrates that one of the most dangerous wine spoilage species, Z. bailii, is strongly associated with sour rot grapes and survives during fermentation with Saccharomyces cerevisiae. The use of selective media provides a more accurate characterization of grape contamination species.

Changes in sour rotten grape berry microbiota during ripening and wine fermentation.

This study investigated the microbiota of sour rotten wine grapes and its impact on wine fermentations. Yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were enumerated and identified on sound and sour rot grapes during the ripening stage. The alteration of the ecological balance induced by sour rot was particularly evidenced by the unequivocal increase of yeast and AAB counts on rotten grapes, since the beginning of ripening. Yeast and AAB species diversity in rotten grape samples were much higher than those found in sound grapes. LAB populations were low detected from both healthy and sour rotten grapes. The yeast species Issatchenkia occidentalis, Zygoascus hellenicus and Zygosaccharomyces bailii and the AAB species Gluconacetobacter hansenii, Gluconacetobacter intermedius and Acetobacter malorum, were recovered from damaged grapes and resulting grape juices in the winery. Acetobacter orleaniensis and Acetobacter syzygii were only recovered from sour rotten grapes. Dekkera bruxellensis and Oenococcus oeni were only recovered after wine fermentation induced by starter inoculation, irrespective of grape health, probably originating from cellar environment. After malolactic fermentation, racking and sulphur dioxide addition the only remaining species were the yeast Trigonopsis cantarellii and Saccharomyces cerevisiae, independently of the grape health status.

Ascomycetous yeast species recovered from grapes damaged by honeydew and sour rot

Aims:  To identify ascomycetous yeasts recovered from sound and damaged grapes by the presence of honeydew or sour rot.