WINETECH Technical Yearbook 2020

oxygen. Increased frequency of aeration interventions, such as punch downs, may enhance the persistence of L. thermotolerans strains (Hansen et al ., 2001; Shekhawat et al ., 2017 & 2018) SUGAR CONSUMPTION AND LACTIC ACID PRODUCTION • Although L. thermotolerans can ferment both glucose and fructose, all strains display a glucophilic character and can produce anything between 0.3 and 16 g/L l -lactic acid, derived from pyruvate in the glycolytic pathway (Benito, 2018; Hranilovic et al ., 2018; Morata et al ., 2020). • The lactic acid is produced in the early stages of fermentation and can lead to reduction in pH by up to 0.5 units. The pH reduction improves wine colour intensity and stability by increasing the molecular SO 2 (Morata et al ., 2020). • Efficient lactic acid production is dependent on L. thermotolerans cell numbers, with a significant effect when the population is above 1 x 10 6 cfu/mL (Morata et al ., 2020). Consequently, fermentation parameters, such as temperature and YAN levels that affect growth, also influence lactic acid production (Morata et al ., 2020). • The diversion of pyruvate to lactic acid can reduce the final ethanol levels in wine by 0.3-1% v/v. The best inoculation

sine have been shown to also sustain viability at the beginning and late stages of the stationary phase (Kemsawasd et al ., 2015). ALCOHOL AND SO 2 TOLERANCE • Lanchancea thermotolerans is a Crab­ tree positive yeast (i.e. it displays a respiro-fermentative metabolism that allows it to ferment sugars even in the presence of excess oxygen). • Strains of L. thermotolerans have been widely described as moderate fermenters, able to produce 5-10% v/v ethanol, but unable to entirely ferment grape must (Morata et al ., 2018; Benito, 2018; Hranilovic et al ., 2018). • Consequently, co-inoculation with a stronger fermenter, such as Saccharo- myces cerevisiae , is necessary in order to ferment grape must to dryness. How- ever, in such mixed-culture fermenta- tions L. thermotolerans strains only persist until the middle of fermentation. • Most strains can tolerate up to 7% v/v ethanol and standard SO 2 levels (25-50 mg/L) typically used in winemaking, while a few show tolerance to higher levels of 8-10% v/v ethanol (Porter et al ., 2019; Morata et al ., 2020). THE ROLE OF OXYGEN The decline of L. thermotolerans during wine fermentation has been attributed to i ts sensi t ivi ty to low dissolved

strategy to achieve efficient pH and ethanol reduction, is shown to be sequential inoculation of S. cerevisiae 48 hours after L. thermotolerans , with an inoculation ratio of 1 x 10 7 cfu/mL L. thermotolerans : 1 x 10 3 cfu/mL S. cerevisiae (Morata et al ., 2018) . IMPACT ON WINE AROMA Apart from the lactic acid production, L. thermotolerans contributes to wine aroma and flavour through fermentation-derived metabolites, such as 2-phenylethanol, 2-phenylethyl acetate and ethyl lactate (Morata et al ., 2018 & 2020; Vilela, 2018). Moreover, L. thermotolerans produces enzymes, such as β-glucosidases and β-lyases, that modulate the release of monoterpenes and thiols and therefore enhance the expression of varietal aromas (Morata et al ., 2018). Overall, L. thermotolerans contributes to the production of wines with enhanced mouthfeel, floral notes, fruitiness and freshness (Vilela, 2018). COMMERCIALLY AVAILABLE PRODUCTS Several L. thermotolerans active dry yeast (ADY) commercial preparations are now available. These include Viniflora ® CON- CERTO™ (CHr Hansen) and Level2 LAK- TIA™ (Lallemand) as monocultures, as well as Viniflora ® Melody™ (CHr Hansen) in mixed cultures with Torulaspora del- brueckii and S. cerevisiae , and Viniflora ®

Rhythm™ (CHr Hansen) in mixed culture with S. cerevisiae. REFERENCES Lachance, M.A. & Kurtzman, C.P. , 2011. Lachancea Kurtzman (2003). In: Kurtzman, C., Fell J.W. & Boekhout, T. (Eds). The yeasts: A taxonomic study, 5th edn. Elsevier, Amsterdam. pp 511-519. Porter, T.J., Divol, B. & Setati, M.E., 2019. Lachancea yeast species: Origin, biochemical characteristics and oenological significance. Food Research International 119, 378-389. Morata, A., Loira, I., Tesfaye, W., Bañuelos, M.A., González, C. & Suárez-Lepe, J.A., 2018. Lachancea thermotolerans appli- cations in wine technology. Fermentation 4, 53. Kemsawasd, V., Viana, T., Ardö, Y. & Arneborg, N., 2015. Influence of nitrogen sources on growth and fermentation performance of different wine yeast-species during alcoholic fermentation. Applied Microbiology and Biotechnology 99, 10191-10207. Roca-Mesa, H., Sendra, S., Mas, A., Beltran, G. & Torija, M-J., 2020. Nitrogen preferences during alcoholic fermentation of different non- Saccharomyces yeasts of oenological interest. Microorganisms 8, 157. Benito, S., 2018. The impacts of Lachancea thermotolerans yeast strains on winema­

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