Screening Non-Saccharomyces Yeasts as Low Ethanol Producing Starter Cultures


Non-Saccharomyces yeasts are known for their low fermentation rate in comparison to Saccharomyces cerevisiae.  In this study, non-Saccharomyces yeasts were inoculated into Chenin blanc grape must and fermented under aerobic and anaerobic conditions.  Saccharomycodes ludwigii displayed a strain-dependent fermentation rate, which yielded between 5.2% and 9.9% ethanol concentration under both conditions, albeit with residual sugar.  Aerobic conditions favoured the production of reduced ethanol which was between 5.8% and 9.7% for non-Saccharomyces yeasts in comparison to S. cerevisiae (10%).  This trend was observed for Wickerhamomyces anomalus, Stamerella bacillaris (Candida zemplinina), Metshnikowia pulcherrima, Cyberlindnera saturnus, Wickerhamomyces subpelliculosus and Cyberlindnera jadinii.  The laboratory-scale wines prepared with the aforementioned yeasts yielded ripe fruit and floral aroma attributes while other non-Saccharomyces yeasts resulted in wines with spicy, acidic and solvent aroma notes.

Author Biographies

N.N. Mehlomakulu

Post-harvest and Agro-processing Technologies Division, ARC Infruitec-Nietvoorbij (The Fruit, Vine and Wine Institute of the Agricultural Research Council), Stellenbosch

J.W. Hoff

Post-harvest and Agro-processing Technologies Division, ARC Infruitec-Nietvoorbij (The Fruit, Vine and Wine Institute of the Agricultural Research Council), Stellenbosch

H. Erten

Department of Food Engineering, Faculty of Agriculture, Cukurova University

L. Beukes

Post-harvest and Agro-processing Technologies Division, ARC Infruitec-Nietvoorbij (The Fruit, Vine and Wine Institute of the Agricultural Research Council), Stellenbosch

N.P. Jolly, Post-harvest and Agro-processing TechnologiesARC Infruitec-NietvoorbijStellenboschSouth Africa

Senor Researcher (Microbiology), Post-harvest and Agro-processing Technologies, ARC Infruitec-Nietvoorbij, Stellenbosch


Bagheri, B., Bauer, F.F. & Setati, M.E., 2015. The diversity and dynamics of indigenous yeast communities in grape must from vineyards employing different agronomic practices and their influence on wine fermentation. S. Afr. J. Enol. Vitic. 36, 243-251.

Canonico, L., Comitini, F., Oro, L. & Ciani, M., 2016. Sequential fermentation with selected immobilized non-Saccharomyces yeast for reduction of ethanol content in wine. Front Microbiol. 7, 278.

Ciani, M. & Comitini, F., 2015. Yeast interactions in multi-starter wine fermentatios. Curr. Opin. Food Sci. 1, 1-6.

Ciani, M., Comitini, F., Mannazzu, I. & Domizio, P., 2010. Controlled mixed culture fermentation: a new perspective on the use of non-Saccharomyces yeasts in winemaking. FEMS Yeast Res. 10, 123-133.

Ciani, M. & Maccarelli, F., 1998. Oenological properties of non-Saccharomyces yeasts associated with wine-making. World J. Microbiol. Biotechnol. 14, 199-203.

Ciani, M., Morales, P., Comitini, F., Tronchoni, J., Canonico, L., Curiel, J.A., Oro, L., Rodrigues, A.J. & Gonzales, R., 2016. Non-conventional yeast species for lowering ethanol content of wines. Front Microbiol. 7, 1-13.

Ciani, M. & Picciotti, G., 1995. The growth kinetics and fermentation behaviour of some non-Saccharomyces yeasts associated with wine-making. Biotechnol. Lett. 17, 1247-1250.

Contreras, A., Hidalgo, C., Schmidt, S., Henschke, P.A., Curtin, C. & Varela, C., 2015. The application of non-Saccharomyces yeast in fermentations with limited aeration as a strategy for the production of wine with reduced alcohol content. Int. J. Food Microbiol. 205, 7-15.

Englezos, V., Rantsiou, K., Cravero, F., Torchio, F., Ortiz-Julien, A., Gerbi, V., Rolle, L. & Cocolin, L., 2016. Starmerella bacillaris and Saccharomyces cerevisiae mixed fermentations to reduce ethanol content in wine. App. Microbial Cell Physiol. 100, 5515-5526.

Erten, H. & Campbell, I., 2001. The production of low-alcohol wines by aerobic yeasts. J. Inst. Brew. 107, 207-215.

Fleet, G.H., 2003. Yeast interactions and wine flavour. Int. J. Food Microbiol. 86, 11-22.

Fleet, G.H., Lafon-Lafourcade, S. & Ribéreau-Gayon, P. 1984. Evolution of yeasts and lactic acid bacteria during fermentation and storage of Bordeaux wines. Appl. Environ. Microbiol. 48, 1034-1038.

García, M., Esteve-Zarzoso, B. & Arroyo, T., 2016. Non-Saccharomyces yeasts: biotechnological role for wine production, In: Morata, A. & Loira, I. (eds). Grape and Wine Biotechnology, InTech. pp. 249-271.

Gonzalez, R., Quiros, M. & Morales, P., 2013. Yeast respiration of sugars by non-Saccharomyces yeast species: A promising and barely explored approach to lowering alcohol content of wines. Trends Food Sci. Technol. 29, 55-61.

Goold, H.D., Kroukamp, H., Williams, T.C., Paulsen, I.T., Varela, C. & Pretorius, I.S., 2017. Yeast's balancing act between ethanol and glycerol production in low-alcohol wines. Microb. Biotechnol. 10, 264-278.

Heard, G.M. & Fleet, G.H., 1987. Occurence and growth of killer yeasts during wine fermentation. Appl. Environ. Microbiol. 53, 2171-2174.

Holm, H.E., Nissen, P., Sommer, P., Nielsen, J.C. & Arneborg, N., 2001. The effect of oxygen on the survival of non-Saccharomyces yeasts during mixed culture fermentations of grape juice with Saccharomyces cerevisiae. J. Appl. Microbiol. 91, 541-547.

Ibrahim R.M., Tariq K.M.A., Khan, S., Awan, M.S. & Hashmi, A.-S., 2004. Production of single cell protein from rice polishings using Candida utilis. World J. Microb. Biotech. 20, 297-301.

Ivit, N.N., Longo, R. & Kemp, B., 2020. The effect of non-Saccharomyces and Saccharomyces non-cerevisiae yeasts on ethanol and glycerol levels in wine. Fermentation 6, 77.

Jolly, N.P., Augustyn, O.P.H. & Pretorius, I.S., 2003. The effect of non-Saccharomyces yeasts on fermentation and wine quality. S. Afr. J. Enol. Vitic. 24, 55-62.

Jolly, N.P., Augustyn, O.P.H. & Pretorius, I.S., 2006. The role and use of non-Saccharomyces yeasts in wine production. S. Afr. J. Enol. Vitic. 27, 15-39.

Jolly, N.P., Varela, C. & Pretorius, I.S., 2014. Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered. FEMS Yeast Res. 14, 215-237.

Lappa, I.K., Kachrimanidou, V., Pateraki, C., Koulougliotis, D., Eriotou, E. & Kopsahelis, N., 2020. Indigenous yeasts: emerging trends and challenges in winemaking. Curr. Opin. Food Sci. 32, 133-143.

Lee, B.-K. & Kim, J.K., 2001. Production of Candida utilis biomass on molasses in different culture types. Aquacult. Eng. 25, 111-124.

Liu, L., Redden, H. & Alper, H.S., 2013. Frontiers of yeast metabolic engineering: diversifying beyond ethanol and glycerol production in low-alcohol wines. Curr. Opin. Biotechnol. 24, 1023-1030.

Liu, P.-T., Lu, L., Duan, C.-Q. & Yan, G.-L., 2016. The contribution of indigenous non-Saccharomyces wine yeast to improved aromatic quality of Cabernet Sauvignon wines by spontaneous fermentation. LWT - Food Sci. Technol. 71, 356-363.

Lõoke, M., Kristjuhan, K. & Kristjuhan, A., 2011. Extraction of genomic DNA from yeasts for PCR – based applications. Biotechniques 50, 325–328.

Lowes, K.F., Shearman, C.A., Payne, J., MacKenzie, D., Archer, D.B., Merry, R.J. & Gasson, M.J., 2000. Prevention of yeast spoilage in feed and food by the yeast mycocin HMK. Appl. Environ. Microbiol. 66, 1066-1076.

Manzanares, P., Vallés, S. & Viana, F., 2011. Non-Saccharomyces yeasts in the wine making process. In: Santiago, C.A.V., Munoz, R. & Garcia, R.G. (eds). Molecular Wine Microbiology. Academic Press, pp. 85-110.

Mehlomakulu, N.N., Setati, M.E. & Divol, B., 2014. Characterization of novel killer toxins secreted by wine-related non-Saccharomyces yeasts and their action on Brettanomyces spp. Int. J. Food Microbiol. 188, 83-91.

O’Donnell, K., 1993. Fusarium and its near relatives. The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics, In: Reynolds, D.R. & Taylor, J.W. (eds), pp. 225-233, CAB International Wallingford, UK.

Palpacelli, V., Ciani, M. & Rosini, G., 1991. Activity of different 'killer' yeasts on strains of yeast species undesirable in the food industry. FEMS Microbiol. Lett. 84, 75-78.

Philliskirk, G. & Young, T.W., 1975. The occurrence of killer character in yeasts of various genera. Antonie Leewenhoek 41, 147-151.

Quiros, M., Rojas, V., Gonzalez, R. & Morales, P., 2014. Selection of non-Saccharomyces yeasts strains for reducing alcohol levels in wine by sugar respiration. Int. J. Food Microbiol. 181, 85-91.

Ribèreau-Gayon, P. & Peynaud E., 1960. Traitè d’Oenologie. Paris et Liège Librarie Polytechnique Ch. Bèranger, Paris, pp. 293-298.

Rodríguez-Cousiño, N., Maqueda, M., Ambrona, J., Zamora, E., Esteban, R. & Ramírez, M., 2011. A new wine Saccharomyces cerevisiae killer toxin (Klus), encoded by a double-stranded RNA virus, with broad antifungal activity is evolutionarily related to a chromosomal host gene. Appl. Environ. Microbiol. 77, 1822-1832.

Sadoudi, M., Tourdot-Maréchal, R., Rousseaux, S., Steyer, D., Gallardo-Chacón, J.-J., Ballester, J., Vichi, S., Guérin-Schneider, R., Caixach, J. & Alexandre, H., 2012. Yeast–yeast interactions revealed by aromatic profile analysis of Sauvignon blanc wine fermented by single or co-culture of non-Saccharomyces and Saccharomyces yeasts. Food Microbiol. 32, 243-253.

Shekhawat, K., Porter, T.J., Bauer, F.F. & Setati, M.E., 2018. Employing oxygen pulses to modulate Lachancea thermotolerans-Saccharomyces cerevisiae Chardonnay fermentations. Ann. Microbiol. 68, 93-102.

Suzzi, G., Romano, P., Ponti, I. & Montuschi, C., 1995. Natural wine yeasts as biocontrol agents. J. Appl. Microbiol. 78, 304-308.

Valente, C.C., Bauer, F.F. Watson, B. & Nieuwoudt, H.H., 2018. Modelling the sensory space of varietal wines: mining of large unstructured text data and visualisation of style patterns. Sci. Rep. 8, 4987.

Van Vuuren, H.J.J. & Jacobs, C.J., 1992. Killer yeasts in the wine industry: A review. Am. J. Vitic. Enol. 43, 119-128.

Varela, C., Contreras, A., Hildago, C., Sengler, F., Schmidt, S., Henschke, P., Chambers, P. & Curtin, C., 2015. Using non-conventional yeast for the production of reduced alcohol wine, Oenoviti International, 4th International Symposium: Non-convential microrganisms in winemaking, University of Padova Conegliano, Italy, 26 October.

Varela, C., Sengler, F., Solomon, M. & Curtin, C., 2016. Volatile flavour profile of reduced alcohol wines fermented with the non-conventional yeast species Metschnikowia pulcherrima and Saccharomyces uvarum. Food Chem. 209, 57-64.

Viana, F., Gil, J.V., Genovés, S., Vallés, S. & Manzanares, P., 2008. Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits. Food Microbiol. 25, 778-785.

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.

Zohre, D.E. & Erten, H., 2002. The influence of Kloeckera apiculata and Candida pulcherrima yeasts on wine fermentation. Process Biochem. 38, 319-324.