Journal of Applied Microbiology 1998, 85, 247–254 Combined use of immobilized Candida stellata cells and Saccharomyces cerevisiae to improve the quality of wines M. Ciani and L. Ferraro Dipartimento di Biologia Vegetale sez. Microbiologia Applicata, Universita ` di Perugia, Perugia, Italy 6480/11/97: received 12 November 1997, revised 9 January 1998 and accepted 13 January 1998 M. CIANI AND L. FERRARO. 1998. Grape must fermentation by the combination of immobilized Candida stellata cells and Saccharomyces cerevisiae was carried out in order to enhance the analytical profiles of wine. Batch and continuous pre-treatment of must with immobilized C. stellata cells, followed by an inoculum of S. cerevisiae, enhanced the analytical profiles of fermentates. The metabolic interactions between the two yeast species showed a positive influence on reducing sugars, acetaldehyde and acetoin metabolism. Sequential fermentation was the best combination for improving the analytical profiles of wine but caused a loss of viability and metabolic activity of beads by limiting their successive use. Continuous pre-treatment of must on the beads of C. stellata could be a more interesting modality to improve the quality of wines. This biotechnological process could be profitably used to produce specific and special wines. INTRODUCTION Natural grape juice fermentation is carried out by a sequence of different yeast species; initially, apiculate yeasts (Kloeckera/ Hanseniaspora) are present but after 3–4 days, these are replaced by elliptical yeasts (Saccharomyces cerevisiae) (Muller-Thurgau 1896). Recent quantitative studies on must fermentation high- lighted the important role of non-Saccharomyces species regarding the analytical composition of wine (Herraiz et al. 1990; Fleet and Heard 1993; Lema et al. 1996). Non- Saccharomyces species, particularly Hanseniaspora uvarum (Kloeckera apiculata) and Candida stellata, survived during the fermentation for longer periods than previously thought (Fleet et al. 1984; Pardo et al. 1989). Moreover, inoculated fermentation with selected yeast cultures belonging to S. cerevisiae species did not necessarily guarantee its dominance (Martinez et al. 1989; Mora and Mulet 1991). Previous studies (Ciani and Picciotti 1995; Ciani and Maccarelli 1998) on metabolic and analytical profiles of some non-Saccharomyces wine yeasts showed that C. stellata could positively affect the taste and flavour of alcoholic beverages. Further studies suggested the use of immobilized C. stellata cells to enhance the glycerol content of wine (Ciani and Ferraro 1996a,b). The aim of this work was to verify the fermentation behaviour of the combination of C. stellata Correspondence to: Dr Maurizio Ciani, Dipartimento di Biologia Vegetale, sez. Microbiologia Applicata, Borgo XX Giugno 74, 06121 Perugia, Italy. © 1998 The Society for Applied Microbiology immobilized cells and S. cerevisiae in grape juice by evaluating the modality of inoculum and the metabolic interactions between the two yeast species. MATERIALS AND METHODS Micro-organisms and media Cultures of C. stellata and S. cerevisiae selected for wine- making were obtained from the Industrial Yeasts Collection of the Dipartimento di Biologia Vegetale, University of Per- ugia (DBVPG). Accession numbers were C. stellata 3827 and S. cerevisiae 6663. All strains were subcultured at 6 month intervals on malt agar medium and maintained at 6 °C. Grape must (cultivar pinot grigio, sugar 18·5% (w/v) pH 3·10) brought to a sugar concentration of 27% (w/v) by addition of sucrose and steam-sterilized at 90 °C for 15 min, was used in the fermentation tests. YPD (Bacto Yeast Extract, 10 g l −1 and Bacto Peptone, 10 g l −1 (Difco), and D-glucose, 50 g l −1 ) was used to produce biomass for the immobilized system. Immobilization Candida stellata cells for immobilization were grown in YPD at 25 °C in a rotary shaker (150 rev min −1 for 72 h, harvested by centrifugation, washed three times with sterile distilled water, and added to 2·5% Na alginate (Carlo Erba, Milan,