Metschnikowia pulcherrima yeast and its role in winemaking

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M. pulcherrima is least influenced by must nitrogen composition compared to S. cerevisiae and other non-Saccharomyces yeasts frequently encountered in wine fermentation.

Current and previous names

Metschnikowia pulcherrima has previously been designated under various names based on classical yeast taxonomy methods. The names include Candida pulcherrima (the anamorph), Eutorula pulcherrima, Monilia castellanii, Torula pulcherrima, Torulopsis pulcherrima, Torulopsis burgeffiana, Torulopsis dattila, Rhodotorula pulcherrima, Saccharomyces pulcherrimus and Cryptococcus castellanii.¹

Where it is found

Metschnikowia pulcherrima is an ubiquitous yeast primarily associated with fruit-feeding insects. However, it has been found on grapes and is commonly isolated from grape juice and grape must. It has also been found in various fresh and spoiled fruit, flowers, nectar and tree sap fluxes.²

What it looks like

Metschnikowia pulcherrima forms ovoid to ellipsoidal cells (2 – 12 µm x 2 – 10 µm). It reproduces asexually by multilateral budding and sexually through the formation of elongate asci containing two needle-shaped spores. The spore formation is preceded by differentiation of diploid cells into thick-walled, refractile, spherical chlamydospores (also referred to as pulcherrima cells) that contain lipid globules. Some strains may form pseudohyphae under anaerobic conditions.^1-3 When cultivated on YPD (yeast extract, peptone and dextrose) agar, M. pulcherrima cells form cream-coloured colonies that produce a reddish-brown soluble pigment (pulcherrimin) visible from the bottom.

Nitrogen metabolism

Metschnikowia pulcherrima can utilise a variety of nitrogen sources. It can use lysine as the sole nitrogen source and can use proline more efficiently than Saccharomyces cerevisiae.⁴ Metschnikowia pulcherrima is one of the non-Saccharomyces yeast species that display low yeast assimilable nitrogen (YAN) uptake and is unable to complete wine fermentation regardless of the nature of the nitrogen source, despite displaying a shorter lag phase on most nitrogen sources. The presence of ammonia in the media does not greatly affect amino acid uptake.^4,5 Overall, M. pulcherrima is least influenced by must nitrogen composition compared to S. cerevisiae and other non-Saccharomyces yeasts frequently encountered in wine fermentation.⁵

Technical characteristics

• Temperature requirements/tolerance

Metschnikowia pulcherrima strains can grow well under low temperature (15 – 20°C), but cannot grow at high temperature (e.g. 37°C). They can also grow over a wide pH range (3 – 6), but stability below pH 4 is low.² Most strains can tolerate up to 40 mg/L total SO₂,^2,6,7 with a few strains including M. pulcherrima Flavia^® MP346 able resistant to higher levels.⁸

• Alcohol tolerance

Most strains of M. pulcherrima can tolerate at least 6% v/v ethanol, with only a few exceptions showing an ability to grow above 9% v/v.⁶

• Oxygen requirements

Metschnikowia pulcherrima displays a highly aerobic respiratory metabolism and requires high levels of oxygen to persist. Indeed, M. pulcherrima falls under the group of non-Saccharomyces yeasts that under suitable aeration conditions more than 40% of the sugar they consume is expected to be respired, whereas in S. cerevisiae only 25% of the sugar is respired.⁹ Furthermore, M. pulcherrima strains show better growth, persistence and sugar consumption under aerobic conditions when in monoculture or in mixed culture fermentations with S. cerevisiae.^10,11

• Glycerol, VA, SO₂ and H₂S production

Metschnikowia pulcherrima generally produces moderate levels (0.3 – 0.4 g/L) of volatile acidity (expressed as acetic acid). When used in sequential fermentation with S. cerevisiae, it is known to reduce VA by 10 – 75% and increase glycerol by 4 – 40%, depending on the inoculation strategy, the strain and the persistence of the strain during fermentation.¹ H₂S production in M. pulcherrima is strain dependent, ranging from absent to high production, with most strains being H₂S negative due to lack of sulphite reductase activity.¹

• Behaviour in pure culture fermentations

Metschnikowia pulcherrima mainly displays low fermentative capacity with most strains producing up to 4.5% (v/v) ethanol. A few strains have been reported to be moderate fermenters able to produce 9 – 11.5% ethanol, albeit over excessively long fermentation periods (e.g. 180 days) in monocultures.^1,12 Consequently, the most preferred and efficient fermentation mode for this yeast is sequential inoculations with S. cerevisiae.^1,2

Effect on malolactic fermentation

Malic acid consumption has been reported in some strains of M. pulcherrima. Regarding influence on malolactic fermentation, several strains have shown compatibility with Oenococcus oeni strains, however, this is dependent on the strain of both the yeast and the bacteria and the inoculation strategy. For instance, wines produced by inoculation of S. cerevisiae at 48 and 72 hours after M. pulcherrima were found to result in slower MLF than when S. cerevisiae was inoculated after 24 hours.¹³

Traits of oenological interest

• Extracellular enzymes – effects on wine aroma/stability

Metschnikowia pulcherrima strains produce a diversity of enzymes that are important in the release of varietal aromas. In particular, M. pulcherrima produces glycosidases, such as β-glucosidase, β-xylosidase and in some strains (e.g. M. pulcherrima Flavia™) α-arabinofuranosidase, which release monoterpenes, mainly linalool and geraniol nerol.^2,8 Furthermore, M. pulcherrima strains produce β-lyase which release volatile thiols. The production of acid proteases is also widely distributed in this species and these enzymes have shown potential to combat haze formation in white wines. Regarding fermentation aromas, M. pulcherrima strains have been shown to consistently increase the production of 2-phenylethanol, fatty acids, acetoin, diacetyl and the total concentration of esters. Among the ethyl esters, ethyl octanoate (a pear-associated ester) is most notable.¹

• Ethanol reduction in wine

Metschnikowia pulcherrima has been proposed as a suitable candidate yeast for lowering the ethanol content in wine. Indeed, several strains when used in sequential inoculation with S. cerevisiae 48 hours after M. pulcherrima or only after 50% of the sugar is consumed and with suitable oxygen input during fermentation were shown to reducing final ethanol levels by 0.6 – 1.6% v/v.^2,14,15 However, this trait is more pronounced in synthetic grape juice fermentation and declines in real wine fermentation conditions, thus further investigation is required.¹⁵

• Chitin/mannoprotein content – effects on wine stability

Studies have shown that some strains of M. pulcherrima display notable mannoprotein release ability which increases the mouthfeel properties of wine.¹⁶ The release of mannoproteins has also been shown to improve protein stability in wine and lower haze potential.¹⁷ Furthermore, recent studies have shown that M. pulcherrima strains including Level 2 Flavia, are able to reduce haze formation even when minimal protease activity is detected. This ability is thought to be due to high levels of chitin in the cell wall. The chitin has been shown to adsorb and subsequently remove grape chitinases which are major contributors to haze.^17,18

Commercial products available and their applications according to suppliers

Only a couple of strains of M. pulcherrima have been commercialised. Amongst them Level 2 Flavia™ and Levulia^® Pulcherrima contribute positively to wine aroma, while Excellence^® B-Nature is mainly used for bioprotection activity and as an alternative to sulphur addition.

References

1. Vicente, J., Ruiz, J., Belda, I., Benito-Vázquez, I., Marquina, D., Calderón, F., Santos, A. & Benito, S., 2020. The genus Metschnikowia in enology. Microorganisms 8, 1038.
2. Morata, A., Loira, I., Escott, C., De Fresno, J.M., Bañuelos, A. & Suárez-Lepe, J.A., 2019. Applications of Metschnikowia pulcherrima in wine biotechnology. Fermentation 5, 63.
3. Lachance, M-A., 2016. Metschnikowia: Half tetrads, a regicide and the fountain of youth. Yeast 33, 563 – 574.
4. Su, Y., Seguinot, P., Sanchez, I., Ortiz-Julien, A., Heras, J.M., Querol, A., Camarasa, C. & Guillamón, J.M., 2020. Nitrogen sources preferences of non-Saccharomyces yeasts to sustain growth and fermentation under winemaking conditions. Food Microbiology 85, 103287.
5. 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.
6. Barbosa, C., Lage, P., Esteves, M., Chambel, L., Mendes-Faia, A. & Mendes-Ferreira, A., 2018. Molecular and phenotypic characterization of Metschnikowia pulcherrima strains from Douro wine region. Fermentation 4, 8.
7. Comitini, F., Gobbi, M., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I. & Ciani, M., 2011. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiology 28, 873 – 882.
8. Morata, A., Escott, C., Bañuelos, A., Loira, I., De Fresno, J.M., González, C. & Suárez-Lepe, J.A., 2020. Contribution of non-Saccharomyces yeasts to wine freshness: A review. Biomolecules 10, 34.
9. Quirós, M., Rojas, V., Gonzalez, R. & Morales, P., 2014. Selection of non-Saccharomyces yeast strains for reducing alcohol levels in wine by sugar respiration. International Journal of Food Microbiology 181, 85 – 91.
10. Shekhawat, K., Bauer, F.F. & Setati, M.E., 2017. Impact of oxygenation on the performance of three non-Saccharomyces yeasts in co-fermentation with Saccharomyces cerevisiae. Applied Microbiology and Biotechnology 101, 2479 – 2491.
11. Morales, P., Rojas, V., Quirós, M. & Gonzalez, R., 2015. The impact of oxygen on the final alcohol content of wine fermented by a mixed starter culture. Applied Microbiology and Biotechnology 99, 3993 – 4003.
12. Du Plessis, H., Du Toit, M., Hoff, J.W., Hart, R.S., Ndimba, B.K. & Jolly, N.P., 2017. Characterisation of non-Saccharomyces yeasts using different methodologies and evaluation of their compatibility with malolactic fermentation. South African Journal of Enology and Viticulture 38, 46 – 63.
13. Martín-García, A., Balmaseda, A., Bordons, A. & Reguant, C., 2020. Effect of the inoculation strategy of non-Saccharomyces yeasts on wine malolactic fermentation. OENO One 54, 1.
14. Hranilovic, A., Gambetta, J.M., Jeffrey, D.W., Grbin, P.R. & Jiranek, V., 2020. Lower-alcohol wines produced by Metschnikowia pulcherrima and Saccharomyces cerevisiae co-fermentations: The effect of sequential inoculation timing. International Journal of Food Microbiology 329, 108651.
15. Puškaš, V.S., Miljić, U., Djuran, J.J. & Vučurović, V.M., 2020. The aptitude of commercial yeast strains lowering the ethanol content of wine. Food Science and Nutrition 8, 1489 – 1498.
16. Belda, I., Navascués, E., Marquina, D., Santos, A., Calderón, F. & Benito, S., 2016. Outlining the influence of non-conventional yeasts in wine ageing over lees. Yeast 33, 329 – 338.
17. Snyman, C., 2019. Impact of the protease-secreting yeast Metschnikowia pulcherrima IWBT Y1123 on wine properties and response of protease production to nitrogen sources. MSc Thesis, Stellenbosch University.
18. Ndlovu, T., Divol, B. & Bauer, F.F., 2018. Yeast cell wall reduces wine haze formation. Applied and Environmental Microbiology 84(13), e00668-18.

– For more information, contact Evodia Setati at setati@sun.ac.za.