Laura Canonico

Non-conventional Yeast Species for Lowering Ethanol Content of Wines.

Rising sugar content in grape must, and the concomitant increase in alcohol levels in wine, are some of the main challenges affecting the winemaking industry nowadays. Among the several alternative solutions currently under study, the use of non-conventional yeasts during fermentation holds good promise for contributing to relieve this problem. Non-Saccharomyces wine yeast species comprise a high number or species, so encompassing a wider physiological diversity than Saccharomyces cerevisiae. Indeed, the current oenological interest of these microorganisms was initially triggered by their potential positive contribution to the sensorial complexity of quality wines, through the production of aroma and other sensory-active compounds. This diversity also involves ethanol yield on sugar, one of the most invariant metabolic traits of S. cerevisiae. This review gathers recent research on non-Saccharomyces yeasts, aiming to produce wines with lower alcohol content than those from pure Saccharomyces starters. Critical aspects discussed include the selection of suitable yeast strains (considering there is a noticeable intra-species diversity for ethanol yield, as shown for other fermentation traits), identification of key environmental parameters influencing ethanol yields (including the use of controlled oxygenation conditions), and managing mixed fermentations, by either the sequential or simultaneous inoculation of S. cerevisiae and non-Saccharomyces starter cultures. The feasibility, at the industrial level, of using non-Saccharomyces yeasts for reducing alcohol levels in wine will require an improved understanding of the metabolism of these alternative yeast species, as well as of the interactions between different yeast starters during the fermentation of grape must.

Screening of yeasts for growth on crude glycerol and optimization of biomass production

Out of 113 yeast strains tested, 45 grew on pure glycerol with growth rates ranging from 0.11 to 0.37h(-1). Twenty-three strains showed specific growth rates (h(-1)), biomass production and biomass yields higher or comparable to those on glucose which suggests that crude glycerol can be utilized as carbon source in yeast cultivation for biomass production. Response surface methodology was applied to optimize crude glycerol concentration and temperature for biomass production and yield by Yarrowia lipolytica (DiSVA C 12.1), Metschnikowia sp. (DiSVA 50), Debaryomyces sp. (DiSVA 45/9), and Rhodotorula mucilaginosa (DiSVA C 7.1). A biomass concentration of 25.7g/l and a biomass yield of 0.92g/g (Y/Xglyc) was obtained with Y. lipolytica DiSVA C 12.1 and with R. mucilaginosa DiSVA C 7.1, respectively. These results demonstrate the potential use of crude glycerol as carbon source in yeast cultivation and the yeast ability to convert low-value crude glycerol to added-value products.

Torulaspora delbrueckii in the brewing process: A new approach to enhance bioflavour and to reduce ethanol content

Nowadays, consumers require fermented alcoholic beverages with particular and enhanced flavour profiles while avoiding the health concerns due to high ethanol content. Here, the use of Torulaspora delbrueckii was evaluated for beer production, in both pure and in mixed cultures with a Saccharomyces cerevisiae starter strain (US-05). The yeast interactions were also evaluated. In mixed fermentations with S. cerevisiae, the main analytical characters from T. delbrueckii were comparable with those of the S. cerevisiae starter strain, but the beers were characterized by a distinctive overall analytical and aromatic profile. Indeed, there were interactions between S. cerevisiae and T. delbrueckii, with enhanced ethyl hexanoate (0.048 mg l(-1)) and ethyl octaonate (0.014 mg l(-1)) levels at the 1:20 and 1:10 inoculation ratios, respectively; while phenyl ethyl acetate increased in all mix combinations. The presence of T. delbrueckii resulted in reduced β-phenyl ethanol and isoamyl acetate levels, which are responsible for floral and fruity aromas, respectively. Beer produced with T. delbrueckii pure cultures had a low alcohol content (2.66%; v/v), while also showing a particularly analytical and aromatic profile.

Yeast interactions in inoculated wine fermentation

The use of selected starter culture is widely diffused in winemaking. In pure fermentation, the ability of inoculated Saccharomyces cerevisiae to suppress the wild microflora is one of the most important feature determining the starter ability to dominate the process. Since the wine is the result of the interaction of several yeast species and strains, many studies are available on the effect of mixed cultures on the final wine quality. In mixed fermentation the interactions between the different yeasts composing the starter culture can led the stability of the final product and the analytical and aromatic profile. In the present review, we will discuss the recent developments regarding yeast interactions in pure and in mixed fermentation, focusing on the influence of interactions on growth and dominance in the process.

Sequential Fermentation with Selected Immobilized Non-Saccharomyces Yeast for Reduction of Ethanol Content in Wine

The average ethanol content of wine has increased over the last two decades. This increase was due to consumer preference, and also to climate change that resulted in increased grape maturity at harvest. In the present study, to reduce ethanol content in wine, a microbiological approach was investigated, using immobilized selected strains of non-Saccharomyces yeasts namely Starmerella bombicola, Metschnikowia pulcherrima, Hanseniaspora osmophila, and Hanseniaspora uvarum to start fermentation, followed by inoculation of free Saccharomyces cerevisiae cells. The immobilization procedures, determining high reaction rates, led a feasible sequential inoculation management avoiding possible contamination under actual winemaking. Under these conditions, the immobilized cells metabolized almost 50% of the sugar in 3 days, while S. cerevisiae inoculation completed all of fermentation. The S. bombicola and M. pulcherrima initial fermentations showed the best reductions in the final ethanol content (1.6 and 1.4% v/v, respectively). Resulting wines did not have any negative fermentation products with the exception of H. uvarum sequential fermentation that showed significant amount of ethyl acetate. On the other hand, there were increases in desirable compounds such as glycerol and succinic acid for S. bombicola, geraniol for M. pulcherrima and isoamyl acetate and isoamyl alcohol for H. osmophila sequential fermentations. The overall results indicated that a promising ethanol reduction could be obtained using sequential fermentation of immobilized selected non-Saccharomyces strains. In this way, a suitable timing of second inoculation and an enhancement of analytical profile of wine were obtained.

Torulaspora delbrueckii contribution in mixed brewing fermentations with different Saccharomyces cerevisiae strains

In recent years, there has been growing demand for distinctive high quality beer. Fermentation management has a fundamental role in beer quality and the levels of aroma compounds. Use of non-conventional yeast has been proposed to enhance beer bioflavor. In the present work we investigated mixed fermentations using three commercial Saccharomyces cerevisiae strains, without and with addition of a selected Torulaspora delbrueckii strain evaluating their interactions, as well as the aroma profiles. At the S. cerevisiae/T. delbrueckii co-inoculation ratio of 1:20, viable cell counts indicated that T. delbrueckii dominated all of the three combinations. In the mixed fermentations, T. delbrueckii provided higher levels of higher alcohols (excepting of β-phenyl ethanol), in contrast to data obtained in winemaking, where higher alcohols had lower levels. Moreover, mixed fermentations showed significantly higher ethyl acetate (from 5 to 16mg/L) and isoamyl acetate (from 0.019 to 0.128mg/L), and were generally lower in ethyl hexanoate and ethyl octanoate. Therefore, irrespective of S. cerevisiae strain, T. delbrueckii influenced on all mixed fermentations. On the other hand, the mixed fermentations were also affected by each of the three S. cerevisiae strains, which resulted in beers with distinctive flavors.

TdPIR minisatellite fingerprinting as a useful new tool for Torulaspora delbrueckii molecular typing.

Torulaspora delbrueckii yeast strains are being increasingly applied at the industrial level, such as in the winemaking process, and so their identification and characterisation require effective, fast, accurate, reproducible and reliable approaches. Therefore, the development of typing techniques that allow discrimination at the strain level will provide an essential tool for those working with T. delbrueckii strains. Here, 28 T. delbrueckii strains from various substrates were characterised using different PCR-fingerprinting molecular methods: random amplified polymorphic DNA with polymerase chain reaction (RAPD-PCR), minisatellites SED1, AGA1, DAN4 and the newly designed T. delbrueckii (Td)PIR, and microsatellites (GAC)5 and (GTG)5. The aim was to determine and compare the efficacies, reproducibilities and discriminating powers of these molecular methods. RAPD-PCR using the M13 primers and the newly designed TdPIR3 minisatellite primer pair provided discrimination of the greatest number of T. delbrueckii strains. TdPIR3 clustered the 28 strains into 16 different groups with an efficiency of 100%, while M13 clustered the strains into 17 different groups, although with a lower efficiency of 89%. Moreover, the TdPIR3 primers showed reproducible profiles when the stringency of the PCR protocol was varied, which highlighted the great robustness of this technique. In contrast, variation of the stringency of the M13 PCR protocol resulted in modification of the amplified profiles, which suggested low reproducibility of this technique.

Occurrence and involvement of yeast biota in ripening of Italian Fossa cheese

A microbiological investigation on a typical Italian Fossa cheese during ripening was reported here. Two yeast isolation campaigns were conducted to investigate the yeast diversity on cheese and pit environment, before and at the end of cheese ripening in pit, using classical and molecular tools. Before the ripening, eight different yeast species were identified from pit environment: Candida zeylanoides, Candida norvegica, Pichia occidentalis, Pichia guilliermondii, Pichia jadinii, Cryptococcus albidus, Cryptococcus skinneri, and Sporobolomyces roseus. Only C. zeylanoides was also found at the end of the cheese-ripening stage, together with the new isolated species Wickerhamomyces anomalus, Saccharomyces cerevisiae, Debaryomyces hansenii, and Candida homilentoma. To evaluate the contributions of these autochthonous species found during ripening, they were inoculated into fresh cheeses. Results show that D. hansenii, C. zeylanoides, and W. anomalus drastically reduced the colonization of molds on the cheese surface, with excellent results of sensory evaluation of the ripened cheese. The cheese inoculated with these indigenous selected yeasts did not show any defects, and volatile organic compounds analysis showed a high concentration of methyl ketones, and butanoic, hexanoic, and octanoic acids, which typically enhance the taste of the highly matured Fossa cheese. These results highlight the positive role of these indigenous yeasts during ripening process of the Fossa cheese.