WINETECH Technical Yearbook 2020

Pichia kluyveri yeast and its role in winemaking

desirable wine aroma outputs in Sauvignon blanc (Anfang et al ., 2009). EFFECT ON MALOLACTIC FERMENTATION Pichia kluyveri strains have not been shown to consume malic acid or produce lactic acid. TRAITS OF OENOLOGICAL INTEREST Generally, P. kluyveri is desired as a co- inoculant in the production of Sauvignon blanc and Riesling due to its ability to enhance thiol production. • Extracellular enzymes Pichia kluyveri produces carbon sul- phur lyases. These enzymes are nec- essary for the release of cysteinylated precursors cys-4MMP and cys-3MH, allowing for the release of the vola- tile thiols 4-mercapto-4-methylpen- tan-2-one (4MMP), 3-mercaptohex- an-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) which are impact odor- ants in Sauvignon blanc wines (Padilla et al ., 2016). Furthermore, P. kluyveri produces esterases and esterase-lipases involved in the release of esters, as well as pectinases, necessary in grape juice processing (Escribano et al ., 2017). • Contribution to wine aroma Pichia kluyveri produces considerable levels of 2-phenylethyl acetate (which

grown in synthetic grape juice (Bedford, 1942; Prior et al ., 2019). The presence of ammonium hampers the uptake of valine, isoleucine and phenylalanine (Prior et al ., 2019). TECHNICAL CHARACTERISTICS • Temperature and alcohol tolerance Pichia kluyveri is a slow grower and a weak fermenter (Prior et al ., 2019). Strains of this species can tolerate 4-5% ethanol (Nutt, 2018) and can grow over a wide temperature range (10-28°C), although at lower temperature a longer lag phase is observed (Nutt, 2018). • Glycerol, VA, SO 2 and H 2 S production Most strains of P. kluyveri produce low levels of H 2 S, less than 0.7 g/L acetic acid and more than 5 g/L glycerol when fermenting grape juice medium with initial sugars at 21°Brix (Mestre Furlani et al ., 2017). • Mixed culture fermentations Pichia kluyveri is considered a weak fermenter and is unable to produce dry wine when used in monoculture (Prior et al ., 2019; Mestre Furlani et al ., 2017; Benito et al ., 2015). Consequently, it is used in sequential inoculation with Saccharomyces cerevisiae. An inoculum ratio of 1:9 ( P. kluyveri : S. cerevisiae ) has been shown to yield

DECEMBER 2020

EVODIA SETATI: Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch KEYWORDS: non- Saccharomyces yeast, volatile thiols, sulphur lyases, esterases

The Pichia kluyveri yeast is desired for its ability to enhance volatile thiols and overall fruitiness in wine. CURRENT AND PREVIOUS NAMES Pichia kluyveri was previously known as Hansenula kluyveri (Jolly et al . 2014) . WHERE IT IS FOUND Pichia kluyveri strains are widely distributed in nature. They have been isolated from grapes, rotting fruit and are commonly encountered in natural fermentations, such as coffee bean and wine fermentations (Jolly et al ., 2003; Brežná et al ., 2010). WHAT IT LOOKS LIKE Pichia kluyveri is known to form ovoid

to elongate cells that may occur singly, in pairs or in short chains, and does not form pseudohyphae. The cells can grow up to 2-6 x 3.5-10 µm. Sexual reproduction leads to the formation of hat-shaped ascospores 1.5-1.75 x 2-2.5 µm in size and 2-4 per ascus (Bedford, 1942). Colonies on Wallerstein Nutrient Agar are cream to mint green in colour, with a rugose texture. NITROGEN METABOLISM Pichia kluyveri grows poorly in synthetic medium and displays low nitrogen consumption in synthetic grape juice (Bedford, 1942; Prior et al ., 2019). Strains of this species do not consume asparagine and glycine and show preferent ial consumption of lysine and arginine when

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