The use of Torulaspora delbrueckii yeast strains for the production of small-scale wines V. Van Breda1,2, N. P. Jolly1* and J. van Wyk2 1Post-Harvest and Wine Technology Division, ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa. *E-mail: jollyn@arc.agric.za 2Department of Food Technology, Cape Peninsula University of Technology, PO Box 1906, Bellville 7535, South Africa. INTRODUCTION Increased local and global competition leading to the ever increasing need for improved wine quality, high alcohols unacceptable to consumers and current resistance by consumers to GMO products are some of the ongoing challenges faced by the South African wine industry. With the current worldwide interest in the positive role played by non‑Saccharomyces yeasts in wine production, the aim of this study was to investigate the potential of commercial and Torulaspora delbrueckii yeast isolates for small-scale Chenin blanc wine production. AIM OF STUDY To investigate the potential of commercial and T. delbrueckii yeast isolates for Chenin blanc production. MATERIALS AND METHODS Yeasts and Yeast inoculum Seven South African T. delbrueckii yeast isolates from the ARC Infruitec-Nietvoorbij yeast culture collection, one commercial T. delbrueckii (TdH) reference strain and one Saccharomyces cerevisiae (Sc1) reference strain were selected for this study based on their performance in laboratory-scale fermentations (van Breda et al., 2013) (Table 1). A three phase propagation procedure was used to grow the yeast in Yeast Peptone Dextrose broth (YPD) (Fig. 1). The T. delbrueckii yeasts were inoculated at a concentration of 2 x 106 cells mL–1 and the S. cerevisiae at a concentration of 1 x 106 cells mL–1. Figure 3: Average fermentation curves of duplicate small‑scale wines of Chenin blanc must fermented at 15ºC. The T. delbrueckii yeast isolates co‑inoculated with the S. cerevisiae (Sc1) reference yeast strain is identified by the time at which the co‑inoculation occurred: (0 h); (24 h); and (48 h). Table 1: Yeasts used in this study. Strain no. Identity Source Year isolated Isolation material Region Sc11 TdH3 206 301 654 704 M2/1 M2/15 M2/27 S. cerevisiae T. delbrueckii na2 nk4 Cabernet Sauvignon must Chardonnay must na nk Stellenbosch Robertson Constantia 1994 1995 1998 1 Commercial S. cerevisiae reference yeast. 2 na ‑ Not applicable, hybrid yeast. 3 Commercial T. delbrueckii reference yeast. 4 nk ‑ Not known. F1 First Principal Component F2 Second Principal Component Figure 4: PCA bi‑plot of average values (n = 2) of sensory analysis descriptors. Figure 1: Preparation of yeast inoculum. Small-scale wine production Small‑scale wine fermentations were conducted in duplicate at 15ºC using a Chenin blanc grape must (22.15ºB, 7.0 g L–1 total acidity, pH 3.42) in 18 L volumes in 20 L stainless steel containers fitted with fermentation caps (Fig. 2). Three T. delbrueckii yeast isolates (strains 654, 301 and M2/1) and one commercial T. delbrueckii yeast strain (TdH), were inoculated individually. Four other T. delbrueckii yeast isolates (strains 704, 206, M2/15 and M2/27) were inoculated in combination with the S. cerevisiae reference strain (Sc1). For the co‑inoculated fermentations, T. delbrueckii was inoculated at 0 h in all cases, followed by inoculation with the S. cerevisiae reference strain at 0 h, 24 h and 48 h, respectively. Fermentations were monitored by CO2 weight loss and continued until dry (residual sugar < 5 g L–1) (data not shown) (Fig. 3) . Those fermentations not completed within 32 d, were stopped. Wines were bottled and stored at 14ºC for four months followed by sensory evaluation and chemical analyses. F1 First Principal Component F2 Second Principal Component Figure 5: PCA bi‑plot of chemical analyses (n = 2) illustrating the yeasts that group together for specific compounds produced. 1 2 3 4 5 6 7 8 9 Figure 2: Small-scale wine production trials at 15ºC. Isolation and identification of yeast from lees Yeast lees samples of the single inoculant wines were cryogenically preserved in glycerol at –80ºC. Selective media was used for single colony isolation which was subjected to ID 32 C (BioMérieux, France) for presumptive identification (data not shown). CHEF gel electrophoreses were then performed using the running conditions as used in van Breda et al., 2013 for final identification. Figure 6: CHEF DNA profiles of the T. delbrueckii isolates in comparison to those isolated from the lees. Lanes 1-9: Sc1; 654 control; Colony 1; Colony 2, Colony 4; Colony 5; Colony 7; Colony 9 and 654 control. CONCLUSION RESULTS & DISCUSSION From the fermentation curves of the small‑scale wines, it was seen that the co‑inoculated fermentations fermented faster (14 d) when compared to the single inoculant fermentations (32 d) (Fig. 3). The S. cerevisiae reference yeast (Sc1) fermented at a faster rate than the T. delbrueckii yeast isolates and conducted the fermentation in 22 d. Three of the fermentations inoculated with the T. delbrueckii yeast isolates only (strains 301, 654 and M2/1), appeared to ferment at a similar rate, while the commercial T. delbrueckii fermentation progressed slightly faster and finished earlier (27 d). Wines were chemically and sensorially evaluated according to the descriptors applicable for Chenin blanc wines. Data was subjected to principle component analysis (Fig. 4 and Fig. 5). The sensory and chemical results of the study showed that non‑Saccharomyces yeasts contributed to the mouthfeel and improved the quality of wines. The DNA profiles of the yeast isolated from the lees samples were compared to that of the yeasts initially inoculated at the start of the fermentation. The profiles appeared similar, indicating that the inoculated yeast were present until the end of fermentation (Fig. 6). Results of this study showed that two T. delbrueckii yeast isolates (strains 654 and M2/1), showed potential for use as single inoculant yeasts in commercial winemaking by producing small‑scale wines that were better in quality to that produced from the reference yeast (Sc1). Strain 654 was also isolated from the lees after fermentation, confirming that the yeast was present throughout the fermentation process and contributed to the relevant fermentation. REFERENCES Van Breda, V.M., Jolly, N.P. & Van Wyk, J. (2013). Characterisation of commercial and natural Torulaspora delbrueckii wine yeast strains. International Journal of Food Microbiology, 163, 80-88. ACKNOWLEDGEMENTS The authors gratefully acknowledge: The financial support by the Agricultural Research Council (ARC), Winetech and NRF-THRIP. ARC Infruitec-Nietvoorbij – The Microbiology Group.