Mixed culture fermentations and Shiraz flavour

by | Aug 1, 2019 | Winetech Technical, Oenology research

PHOTO: Shutterstock.

This study follows similar studies done by us on Chenin blanc, Chardonnay and Pinotage. The aim of this study was to determine the effect of seven different non-Saccharomyces yeast strains in combination with Saccharomyces cerevisiae and three malolactic fermentation strategies had on Shiraz flavour.



Shiraz, also known as Syrah (Vitis vinifera L.), is renowned for “spicy”, “dark fruit”- and “berry”-like flavours and different wine styles can be produced, depending on the region of origin, as well as viticultural and winemaking practices.1

Non-Saccharomyces yeasts possess different oenological properties to those of the standard wine yeast, Saccharomyces cerevisiae, and can be used to modulate and improve the wine aroma and complexity.2 Most non-Saccharomyces yeasts are poor fermenters and are therefore used in combination with S. cerevisiae in co-inoculations, to complete the fermentation.

Malolactic fermentation (MLF) is an enzymatic reaction performed by lactic acid bacteria (LAB) and is required for the production of some red wines, full-bodied white and sparkling wines.3 Malolactic fermentation increases microbiological stability of wine and can affect wine flavour through the modification or production of compounds, such as diacetyl, esters, higher alcohols and volatile acids.4 Various MLF strategies have been investigated with simultaneous (at the start of alcoholic fermentation) and sequential inoculation (after alcoholic fermentation) receiving the most attention.3 Selecting compatible yeast and LAB strains are essential for successful alcoholic and malolactic fermentation, as it has been shown that certain yeast strains negatively affect LAB growth and MLF.5

With the increased use of non-Saccharomyces yeasts, a need exists for a clearer understanding of the interactions that occur between S. cerevisiae, non-Saccharomyces yeasts and LAB. In a previous study,6 37 non-Saccharomyces yeast strains were evaluated for use in wine production. The current study investigated the use of seven non-Saccharomyces strains, from five different species, in combination with S. cerevisiae and three MLF strategies.


Materials and methods

Each of the following seven non-Saccharomyces yeast strains, namely

  • Candida zemplinina (synonym Starmerella bacillaris) (1),
  • Lachancea thermotolerans (2),
  • Metschnikowia pulcherrima (1),
  • Hanseniaspora uvarum (1) and
  • Torulaspora delbrueckii (2),

were used in mixed culture fermentations in combination with one S. cerevisiae wine strain. Shiraz wines produced with S. cerevisiae on its own with or without MLF, served as the reference treatments. Yeast strains were commercially available cultures or were obtained from the ARC Infruitec-Nietvoorbij microorganism culture collection. Each of the eight yeast treatments were also evaluated in combination with the three MLF strategies (no MLF, co-inoculation and sequential inoculation). For co-inoculation, LAB were inoculated 24 hours after yeast and for sequential inoculation, LAB were added after alcoholic fermentation. In total, 24 treatments were evaluated in triplicate. A commercial Oenococcus oeni culture was used to induce MLF. A standardised small-scale (20 L) winemaking protocol was followed at an ambient temperature of 24°C. After completion of MLF, wines were bottled and stored at 15°C. Wines were subjected to descriptive sensory evaluations 24 months later.


Results and discussion

All the Shiraz wines fermented to dryness (residual sugar ˂4 g/L) within 18 days. In most cases, wines produced with non-Saccharomyces yeast had lower alcohol levels (15.49 – 15.94% v/v) than wines produced with S. cerevisiae only (~16% v/v).

Wines produced with C. zemplinina in combination with S. cerevisiae contained the lowest alcohol levels (15.49% v/v). Wines produced with non-Saccharomyces yeasts had higher VA levels than wines produced with S. cerevisiae only, but were well below the legal limits. The difference in VA levels was not detected by the sensory panel.

Results show clearly that there were differences among the non-Saccharomyces strains with regard to their effect on LAB growth and progress of MLF. In most cases, wines produced with non-Saccharomyces yeasts completed MLF in a shorter period than wines produced with only S. cerevisiae. H. uvarum in combination with S. cerevisiae was most compatible with the inoculated LAB and progress of MLF. The C. zemplinina and one of the L. thermotolerans strains had a negative effect on LAB growth and therefore MLF in the co-inoculated MLF wines. However, the same effect was not observed with the yeasts when the MLF culture was sequentially inoculated. The inhibitory effect could be due to competition for essential nutrients or production of toxic metabolites.

Wines produced with the eight yeast combinations and the three MLF strategies (no MLF, co- and sequential inoculation) resulted in all 24 treatments producing wines with significantly different volatile chemical profiles. The sensory data show that the different yeast combinations had a significant effect on the berry aroma of wines, while MLF strategy (no MLF, co- and sequential inoculation) had a significant effect on berry aroma, acid balance and astringency. Wines with co-inoculated MLF scored slightly higher for berry aroma than wines without MLF, but both treatments scored significantly higher than wines with sequential MLF. With regard to acid balance, sequential MLF wines were perceived to be less balanced. However, this did not negatively affect the perceived quality of these wines, because they scored similar or better for overall quality than wines without MLF. Co-inoculated MLF wines were perceived to be more astringent than wines without MLF and significantly more astringent than sequential MLF wines. None of the treatments produced wines that were considered unacceptable with regard to astringency.

Wines produced with non-Saccharomyces yeasts were different to the S. cerevisiae reference wines. The non-Saccharomyces yeast isolates showed potential for producing wines with different styles and flavour profiles, but need further evaluation with different grape cultivars/varieties and in combination with different S. cerevisiae yeast strains. The yeast treatment and the stage of MLF induction had a significant effect on the standard chemical parameters and volatile composition of the wines, although the variation in wine composition did not always translate to perceivable sensory differences.



Results indicated that most non-Saccharomyces yeast strains investigated had a beneficial effect on the progress of MLF. Therefore, if MLF is required, it is important to choose Saccharomyces and non-Saccharomyces strains that are compatible and promote MLF. On the contrary, spontaneous and inoculated MLF can be delayed if yeast strains or combinations are used that negatively affect LAB growth. Selecting suitable non-Saccharomyces yeasts can enhance complexity and produce wines with lower alcohol levels. Wine flavour profiles can be modified by using different non-Saccharomyces yeast strains and MLF strategies. Induction of MLF as a co- or sequential inoculation can also result in significant changes to wine flavour profiles. The interactions between Saccharomyces, non-Saccharomyces and LAB are complex and the resulting changes to wine composition and flavour need further investigation.



The use of non-Saccharomyces yeasts in combination with lactic acid bacteria (LAB) to improve the complexity and diversity of wine styles has received limited attention. Therefore, this study compared the interactions between seven non-Saccharomyces yeast strains and Saccharomyces cerevisiae in combination with three malolactic fermentation (MLF) strategies in a Shiraz winemaking trial. Standard oenological parameters, volatile composition and sensory attributes were investigated. Wines produced with non-Saccharomyces yeasts had lower alcohol and glycerol levels than the reference wines produced with only Saccharomyces cerevisiae, and the duration of malolactic fermentation was also shorter. Non-Saccharomyces wines also differed chemically and sensorially from wines produced with only S. cerevisiae. The Candida zemplinina and one of the Lachancea thermotolerans isolates slightly inhibited LAB growth in wines that underwent simultaneous MLF. Generally, MLF strategy had a greater impact on wine sensory attributes than yeast treatment. Both yeast selection and MLF strategy had a significant effect on berry aroma of wines, but MLF strategy also had a significant effect on acid balance and the perceived astringency. By selecting and applying the correct yeast combination and MLF strategy winemakers can ensure rapid completion of MLF and improved wine complexity.



The authors thank the Agricultural Research Council, Winetech and the National Research Foundation of South Africa (THRIP programme; grant numbers UID 71526 and 90103) for funding, and Justin Hoff, Chrizaan du Plessis, Philda Adonis, Sonia Podgorski and Robyn-Lee Louw are thanked for technical assistance. The opinions, findings and conclusions expressed in this publication are those of the authors. The National Research Foundation accepts no liability in this regard.



  1. Mayr, C.M., Geue, J.P., Holt, H.E., Pearson, W.P., Jeffery, D.W. & Francis, I.L., 2014. Characterization of the key aroma compounds in Shiraz wine by quantitation, aroma reconstitution, and omission studies. Journal of Agricultural and Food Chemistry 62, 4528 – 4536.
  2. Jolly, N.P., Varela, C. & Pretorius, I.S., 2014. Not your ordinary yeast: Non-Saccharomyces yeasts in wine production uncovered. FEMS Yeast Research 14, 215 – 237.
  3. Bartowsky, E.J., Costello, P.J. & Chambers, P.J., 2015. Emerging trends in the application of malolactic fermentation. Australian Journal of Grape and Wine Research 21, 663 – 669.
  4. Swiegers, J.H., Bartowsky, E.J., Henschke, P.A. & Pretorius, I.S., 2005. Yeast and bacterial modulation of wine aroma and flavor. Australian Journal of Grape and Wine Research 11, 139 – 173.
  5. Alexandre, H., Costello, P.J., Remize, F., Guzzo, J. & Guilloux-Benatier, M., 2004. Saccharomyces cerevisiae-Oenococcus oeni interactions in wine: Current knowledge and perspectives. International Journal of Food Microbiology 93, 141 – 154.
  6. Du Plessis, H.W., 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.


Authors: Heinrich du Plessis, Maret du Toit, Hélène Nieuwoudt, Marieta van der Rijst, Martin Kidd & Neil Jolly

– For more information, contact Heinrich du Plessis at dplessishe@arc.agric.za.



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