Evaluation of the Yeast Schizosaccharomyces japonicus for Use in Wine Production

Transcription

1 Evaluation of the Yeast Schizosaccharomyces japonicus for Use in Wine Production Paola Domizio, 1,2 * Livio Lencioni, 2 Luca Calamai, 3 Lorenzo Portaro, 2 and Linda F. Bisson 1 * Abstract: Schizosaccharomyces japonicus UCD2489 was evaluated for potential use as a starter culture in winemaking. Laboratory-scale fermentations of Trebbiano grape juice were set up to compare fermentation kinetics of pure cultures of Sch. japonicus, immobilized Sch. japonicus cells, a commercial strain of Saccharomyces cerevisiae (EC1118), and mixed cultures of both species. The fermentation kinetics of the sequential and coinoculated fermentations were largely driven by the presence of S. cerevisiae. UCD2489 cell immobilization resulted in a significant reduction in ethanol levels in mixed fermentations compared with EC1118. Acetic acid levels similar to those of EC1118 in pure culture were produced when fermentations were coinoculated. The ability of UCD2489 to consume malic acid was adversely affected by EC1118, particularly in the coinoculated fermentation, suggesting that acid levels could be manipulated by adjusting the relative ratios of the two yeasts and the timing of inoculation with S. cerevisiae. Depending upon the inoculation conditions used, Sch. japonicus produced a quantity of glycerol ~2-fold higher than those released by S. cerevisiae. The analyses of volatile compounds showed increases in aroma-impacting compounds such as ethyl acetate in all Sch. japonicus wines, and acetaldehyde in the free-cell coinoculated fermentation that exceed reported sensory thresholds for these compounds, and for other important aroma compounds such as isoamyl acetate, hexyl acetate, phenyl ethyl acetate, ethyl isobutyrate, and ethyl butyrate. Polysaccharide release by UCD2489 was ~4.7-fold greater than that of S. cerevisiae alone. Reduction of induced wine protein haze was correlated with the concentration of polysaccharides. Our findings suggest that Sch. japonicus could be useful in wine production to reduce acidity and final ethanol levels and to increase glycerol, volatile compounds, and active polysaccharides with potential beneficial enhancement of protein stability. Key words: mannoprotein, mixed fermentation, non-saccharomyces, polysaccharide, Schizosaccharomyces japonicus, wine, yeast Yeasts belonging to the genus Schizosaccharomyces have been proposed for use in wine fermentation because of their ability to metabolize malic acid (Rankine 1966, Ciani 1995, Silva et al. 2003), permitting nonbacterial biological deacidification and averting production of amines (Benito et al. 2015). These yeasts have also been shown to reduce gluconic acid (Peinado et al. 2004, 2007, 2009) and ethyl carbamate, and to produce pyruvic acid in wine (Benito et al. 2012, 2014). Yeasts belonging to the Schizosaccharomyces genus are also able to release high quantities of polysaccharides into the media (Domizio et al. 2017). In that study, three different strains of Schizosaccharomyces japonicus were characterized, and all showed a higher release of polysaccharides than 1 Department of Viticulture and Enology, University of California-Davis, Davis, CA 95616; 2 Dipartimento di Gestione Sistemi Agrari, Alimentari e Forestali (GESAAF), Università degli Studi di Firenze, Firenze, Italy; and 3 Dipartimento di Scienze Produzioni Agroalimentari e dell Ambiente (DISPAA) Piazzale delle Cascine, 28 Università degli Studi di Firenze, Firenze, Italy. *Corresponding authors (paola.domizio@unifi.it, tel: ; lfbisson@ucdavis.edu, tel: ) Supplemental data is freely available with the online version of this article at Manuscript submitted Jan 2018, revised Mar 2018, accepted Mar 2018 Copyright 2018 by the American Society for Enology and Viticulture. All rights reserved. doi: /ajev Schizosaccharomyces pombe yeast strains. The polysaccharides released were identified as galactomannoproteins. In general, yeast polysaccharides have been reported to have many positive effects on wine quality such as reducing protein and tartrate instability (Lubbers et al. 1993, Waters et al. 1994, Moine-Ledoux and Dubourdieu 1999, Dupin et al. 2000, Brown et al. 2007, Gonzalez-Ramos et al. 2008), increasing the fullness sensation (Vidal et al. 2004), interacting with polyphenols aggregates and consequently smoothing the astringency perception (Escot et al. 2001, Poncet-Legrand et al. 2007, Quijada-Morín et al. 2014), and retaining wine aroma compounds (Lubbers et al. 1994, Chalier et al. 2007). However, Schizosaccharomyces yeasts have also been associated with the presence of off-characters when left too long in the wine after maloalcoholic fermentation (Ciani et al. 2010). To overcome the problem of off-character production, cells of Sch. pombe have been immobilized in calcium alginate, used in mixed fermentation with Saccharomyces yeasts, and removed once the desired malic level is achieved (Magyar and Panyik 1989, Silva et al. 2003, Portugal et al. 2011). To date, yeasts belonging to the species Sch. japonicus have not been extensively evaluated for use in winemaking. This species has many properties similar to those of Sch. pombe and therefore possesses the potential to produce the same or even enhanced benefits, but it may also present similar challenges. Previous studies have used a synthetic grape juice to define the metabolic characteristics of Sch. japonicus (Domizio et 266

2 Schizosaccharomyces japonicus in Wine Production 267 al. 2017). In this study, we extend the characterization of Sch. japonicus to natural grape juice fermentation and evaluate its potential for use as a wine starter culture in mixed fermentation with Saccharomyces. Materials and Methods Yeast strains. A yeast strain belonging to the species Sch. japonicus (UCD2489) from the yeast culture collection of the Department of Viticulture and Enology University of California, Davis, was used. A commercial strain, Lalvin EC1118 (Lallemand Inc.), was used as reference strain for Saccharomyces cerevisiae and for comparison determinations. Evaluation of yeast characters on agar plates. Killer factor and hydrogen sulfide (H 2 S) production by Sch. japonicus were tested on agar plates. Killer character was evaluated using the plate assay described by Rosini (1985), with positive killer activity defined as inhibition of growth of a sensitive strain (S. cerevisiae DBVPG 6500), and detected as a clear zone surrounding the seeded strain. H 2 S production potential was determined by plating the yeasts onto Biggy Agar (Oxoid Unipath Ltd.). On this medium, H 2 S-positive strains generate brown or black colonies rather than the cream to white colonies typical of H 2 S-negative strains. Colony color was evaluated after 48 to 72 hrs of incubation at 26 C. The DB- VPG 1883 wine strain of S. cerevisiae (an H 2 S nonproducing strain) was used as negative control. Fermentation trials. The fermentations were conducted at 22 C in 500 ml Erlenmeyer flasks containing 350 ml Trebbiano grape juice. To evaluate the specific impact of the UCD2489 strain Sch. japonicus on wine compared with that of S. cerevisiae, the grape juice was pasteurized (15 min at 80 C), avoiding the interference of metabolic activity or of by-products produced by yeasts or bacteria naturally present in nonpasteurized grape juice. The pasteurized grape must had the following composition: ph 3.2; reducing sugars, 24% (w/v); titratable acidity, 8.9 g/l, as tartaric acid; malic acid, 2.8 g/l; ammonia, mg N/L; primary amino nitrogen, 79.6 mg N/L; total SO 2, 20 mg/l. Fermentations were carried out in triplicate, according to the scheme shown in Table 1. The flasks were inoculated with precultures grown at 25 C for 48 hrs in the same grape must used for the fermentation trials. The volumes to be inoculated were determined on the basis of cell counting under a light microscope with a Thoma-Zeiss chamber. Pure-culture fermentations of S. cerevisiae were carried out as controls. For the immobilization of the yeast Sch. japonicus, the cells were centrifuged, washed with sterile water, and 5% (wet weight) of cells were then immobilized in alginate beads. The flasks were equipped with Müller valves containing sulfuric acid, allowing the CO 2 to escape. The flasks were continuously agitated at 150 rpm and weighed every day until the end of the fermentation (defined as a constant weight for two consecutive days) to monitor the fermentation kinetics. During the fermentation, samples were taken for the analytical determinations and viable cell counts. Yeast immobilization in alginate beads. Sch. japonicus cells, pregrown at 25 C for 48 hrs, were harvested by centrifugation (6000g 10 min), washed two times with sterile water, and immobilized in alginate according to the procedure described by Rosini and Ciani (1993), with some modifications. In particular, 5% of cells (wet weight) were suspended in 2.5% (w/v) sodium alginate (Sigma-Aldrich). This suspension was then added dropwise by a peristaltic pump (~4 ml/min) to a precipitation bath, containing a calcium chloride (CaCl 2 ) solution (5 g/l). This gave beads with diameters of ~4 mm. The calcium alginate beads were left hardening overnight in the CaCl 2 solution. After that, to eliminate all salt content, the beads were washed with deionized water several times until the conductivity of the washing water was close to that of the deionized water. Then, 35 g (wet weight) of beads were added to 500 ml Erlenmeyer flasks containing 350 ml of grape juice. These 35 g contained ~ total cells, based on the cell count of the centrifuged biomass, determined using a light microscope and a Thoma-Zeiss chamber. The final cell concentration in the alginate was confirmed by plating a sample of the alginate cell suspension (before dropping and hardening it in the CaCl 2 solution and after an appropriate dilution) onto petri dishes containing yeast extract-peptone-dextrose (YPD) agar (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose, and 20 g/l agar, ph 6.5) to confirm that the viable count was consistent with the projected total cell count. Viable cell count. Samples were taken from each flask during the alcoholic fermentation, and the dilution series was plated onto YPD plates. S. cerevisiae and Sch. japonicus display distinctly different colony morphologies on this medium, and it was therefore possible to accurately quantify the cell concentration of S. cerevisiae by direct plating. In contrast, it was not possible to similarly measure the growth kinetics of Sch. japonicus because this strain flocculated after the second day of fermentation, making an accurate assessment of viable cell count on plate agar difficult. Code Table 1 Experimental scheme of the fermentation conditions used. Yeast species (inoculum form) Inoculum cell concn (cells/ml) Inoculation modality a SCF Saccharomyces cerevisiae 10 7 Pure culture SZF SZI SZF + SCF SZI + SCF SZI + SCF 48 hr Schizosaccharomyces japonicus (free cells) Sch. japonicus (immobilized cells) Sch. japonicus (free cells) S. cerevisiae Sch. japonicus (immobilized cells) S. cerevisiae Sch. japonicus (immobilized cells) S. cerevisiae (inoculated after 48 hrs) 10 6 Pure culture 10 7 Pure culture 10 6 Coculture Coculture Sequential culture a Coculture: S. cerevisiae and Sch. japonicus inoculated at the same time; sequential culture: S cerevisiae inoculated 48 hrs after Sch. japonicus.

3 268 Domizio et al. Analytical determinations of fermentation products. Ethanol, residual sugars, and organic acids were determined by high-performance liquid chromatography (HPLC). Twenty µl of the filtered samples (0.45 µm nitrocellulose membranes) were then injected into the HPLC apparatus (Varian Inc., equipped with a 410 series autosampler, a 210 series pump, a 356-LC refractive index detector, and a 335-LC diode array detector, set at 210 nm). Isocratic separation was performed at 75 C on a (( ) cm 7.8 mm) Phenomenex column (Rezex-ROA Organic Acids, sulfonated styrene divinyl-benzene matrix in H+ form). The mobile phase was 10.5 mm sulfuric acid (H 2 SO 4 ) at a flow rate of 0.6 ml/min. Each compound was quantified by comparison with its relevant external calibration curve (from 0.5 g/l to 20 g/l), and the areas of the related peaks were recorded and integrated using Galaxie Chromatography Data System version (Varian Inc.). Using the same HPLC apparatus, the total polysaccharide contents were also evaluated according to Domizio et al. (2014). Isocratic separation of the polysaccharides was performed at 65 C on a Supelco TSK G-OLIGO-PW (808031) column (30 cm 7.8 mm i.d.) equipped with a Supelco TSK- GEL OLIGO (808034) guard column (4 cm 6 mm i.d.). A mobile phase of 0.2 M sodium chloride (NaCl) at a flow rate of 0.8 ml/min was used. Quantification was performed by comparison with an external calibration curve of mannans from S. cerevisiae (M7504, Sigma-Aldrich), at concentrations ranging from 50 mg/l to 500 mg/l, and the area of the mannans peak was recorded and integrated by using the same software as above. For each wine, the values are reported as increments with respect to the initial amounts of total polysaccharides in the must. All analyses were carried out in duplicate. Ammonia nitrogen and primary amino nitrogen were determined using an enzymatic method (Kit K-LARGE and K- PANOPA, respectively, Megazyme). All analyses were carried out in duplicate. Volatile compound analysis. The solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC- MS) technique was used to measure the major volatile compounds associated with yeast activity. An Agilent 7820 GC chromatograph equipped with a 5977 MSD with electron ionization was used for GC-MS analysis. A three-phase Carboxen/PDMS/DVB 75 µm SPME fiber (Supelco) was exposed in the head space of the vials at 60 C for 30 min for volatile compound sampling after 5 min equilibration time. A Gerstel MPS2 XL autosampler equipped with a magnetic transportation adapter and a temperature-controlled, agitated tray was used for ensuring consistent SPME extraction conditions. The volatile compounds were sampled by a Supelco Carboxen/PDMS/DVB 75 µm, 1-cm-long StableFlex fiber. Chromatographic conditions were the following: the column was a J&W Innovax 50 m, 0.2 mm, and i.d. 0.4 µm DF and the injection temperature was 250 C, a splitless mode was used, and the oven temperature started at 40 C for 1 min and then increased at 2 C/min to 60 C, 3 C/min to 150 C, 10 C/min to 200 C, and 25 C/min to 260 C, held for 6.6 min. Mass spectra were acquired within the m/z interval of 29 to 350 in scan mode at a scan speed adjusted to obtain five scans per second. As the SPME technique is susceptible to sample complexity and fiber wearing, an internal standard mixture was prepared and added to the samples, for use in normalization of the analyte area. On the basis of the compounds present in fermented liquids, the following compounds were selected: ethyl acetate-d8; butanol-d10; ethyl hexanoate-d11; acetic acid-d3; hexanoic acid-d11; 3,4 dimethyl phenol; and 5-methylhexanol. These compounds were either deuterated analogs of compounds present in the samples (isotopologues) or compounds with similar chemical properties, but not present in the specimens. The peak areas of these compounds were used for normalizing the peak areas of all quantified compounds according to their chemical properties, elution order, or both. The selection of the most suitable internal standard for each analyte was done as described (Fortini et al. 2017). Wine protein haze tests. At the end of the alcoholic fermentation, protein stability was assessed by determining the induced haze value by two independent methods: Proteotest (Vason) and Bentotest (Richard Wagner), according to the manufacturer s instructions. The Proteotest kit uses the reaction between the protein in the wine and a phenolics-based material. Bentotest is a commercial solution of phosphomolybdic acid in hydrochloric acid and induces haze via protein denaturation. The turbidity of wines after treatments was determined with a nephelometer (HI88703 turbidimeter, Hanna Instrument Inc.). Data analysis. The data were analyzed using the Statgraphics Plus software (version 2.1, for Windows). To evaluate the statistical significance of the chemical data of the wines, the data were subjected to one-way analysis of variance (ANOVA, general linear model). The differences between data were tested with Tukey s honest significant difference test at the 0.05 significance level. The means and the standard error of the mean (mean ± SE) are also reported. Results Cell growth and fermentation performance. The growth kinetics of the S. cerevisiae strain during pure and mixed fermentations at laboratory scale (Figure 1A, 1D, 1E, and 1F) were evaluated to determine the impact of inoculation practice on mixed-culture fermentations with free or immobilized cells of the UCD2489 strain Sch. japonicus. Sch. japonicus cells flocculated after the second day of alcoholic fermentation; as a consequence, it was not possible to measure precisely its growth kinetics after the flocculation. In the coinoculated fermentations (SZF + SCF and SZI + SCF), the presence of Sch. japonicus did not have evident effects on the growth of S. cerevisiae, which showed growth kinetics similar to those of the pure S. cerevisiae culture (SCF). In agreement, the fermentation kinetics (as CO 2 production) of the pure culture of S. cerevisiae (Figure 1A) were comparable to those of the coinoculated fermentations (SZF + SCF and SZI + SCF) (Figure 1D and 1E). In contrast, a lower fermentation rate was observed during the first four days in the sequential fermentation trials (SZI

4 Schizosaccharomyces japonicus in Wine Production SCF 48 hr) (Figure 1F). Despite this, a similar quantity of CO 2 was produced at the end of fermentation, compared to the control fermentation with S. cerevisiae strain (SCF) (9.54% and 10.1% [w/v], respectively). To exclude possible killer toxin activity of Sch. japonicus against S. cerevisiae EC1118, we carried out tests as described (Rosini 1985), and observed no inhibition (data not shown). After three days of fermentation, the pure culture of Sch. japonicus (both in immobilized and free form) utilized just 29% of ammonia and 68 to 70% of primary amino nitrogen, compared with 99% and 92%, respectively, by the pure culture of S. cerevisiae (Figure 2A and 2B). At the end of the fermentation, a high quantity of ammonia (76 to 81 mg N/L) was still present in the Sch. japonicus pure-culture fermentations Figure 1 Growth kinetics of Saccharomyces cerevisiae (dashed lines) and fermentation kinetics (as CO 2 production) of pure and mixed cultures (solid lines). Left panels show pure cultures, and right panels show mixed cultures. (A) Pure cultures of S. cerevisiae (SCF), (B) pure cultures of free cells of Schizosaccharomyces japonicus (SZF), (C) pure cultures of immobilized cells of Sch. japonicus (SZI), (D) S. cerevisiae in mixed culture with free cells of Sch. japonicus (SZF + SCF), (E) S. cerevisiae in mixed culture with immobilized cells of Sch. japonicus (SZI + SCF), and (F) S. cerevisiae in sequential cultures with immobilized Sch. japonicus cells (SZI + SCF 48 hr). Data represent three independent experiments. Error bars show standard deviations of three independent experiments.

5 270 Domizio et al. Figure 2 Kinetics of ammonia (A) and free amino nitrogen consumption (B) at three days, six days, and 14 days of alcoholic fermentations with different inoculation treatments. Data represent three independent experiments. Error bars show standard deviations of three independent experiments. See Table 1 for definitions of x-coordinates. (SZF and SZI). After a quick consumption of primary amino nitrogen during the first six days of fermentation, an increase in primary amino nitrogen was observed at the end of all mixed fermentations (Figure 2B). Stability of immobilization. During, as well as at the end of, the alcoholic fermentation, the release of Sch. japonicus cells from the alginate beads was checked by microscopic observation. After three to four days of fermentation, few asci, each containing eight spores, were observed in the media, suggesting that some Sch. japonicus cells released from the immobilization sporulated in the fermenting juice. Since spores remain in a dormant state until more favorable conditions appear, they would not be expected to grow in subsequent vinification phases in which permissive conditions are not expected to occur. Metabolic profile of fermentations. The low fermentative activity of the Sch. japonicus strain resulted in a significant reduction in ethanol levels in mixed fermentations with immobilized Sch. japonicus cells, both in coinoculation mode (SZI + SCF) and sequential mode (SZI + SCF 48 hr), compared with the S. cerevisiae pure culture fermentation (SCF), (total ethanol content 14.4% [v/v]) (Table 2). In contrast, ethanol values similar to those in the S. cerevisiae pure culture (SCF) were obtained in the coinoculated fermentation with nonimmobilized Sch. japonicus (SZF + SCF). Despite the low quantity of fermented sugar, the pure culture of Sch. japonicus produced a high quantity of volatile acidity when inoculated in the free form (0.76 g/l), but not when immobilized (0.34 g/l) (Table 2). Levels of volatile acidity comparable to those in the S. cerevisiae pure culture (0.28 g/l) were observed when Sch. japonicus cells, both free and immobilized, were coinoculated (SZF + SCF and SZI + SCF) (0.29 to 0.38 g/l, respectively). In contrast, a significant increase in acetic acid production was observed for the sequentially cultured wine (SZI + SCF 48 hr), although it remained ~0.6 g/l (Table 2). The ability to metabolize malic acid, typical of yeast belonging to Sch. pombe species, is here confirmed also for Sch. japonicus (Table 3). In particular, at day three of the alcoholic fermentation, Sch. japonicus strain UCD2489, both in free and in immobilized form (SZF and SZI, respectively) converted 52% and 59% of l-malic acid, respectively. Further malic acid consumption was observed until the end of the fermentation, reaching 73% and 83% of malic acid degradation. In the sequential fermentation (SZI + SCF 48 hr), 58% of l-malic acid was fermented within the first three days of fermentation, compared with 11% and 26% in the coinoculated conditions (SZF + SCF and SZI + SCF, respectively). Interestingly, Sch. japonicus (both immobilized and free forms) produced levels of glycerol (15.94 and 14.71, respectively) that were ~2-fold greater than that produced by S. cerevisiae (7.38 g/l) at the end of grape juice fermentation (Table 2). The mixed sequential fermentation trial (SZI + SCF 48 hr) yielded amounts of glycerol (12.54 g/l) similar to those produced by Sch. japonicus in pure culture (SZF and SZI). In contrast, in the coinoculations (SZF + SCF and SZI + SCF), Table 2 Main enological parameters of wines after 14 days of alcoholic fermentation. a Variable b SCF c SZF c SZI c SZF + SCF c SZI + SCF c SZI + SCF 48 hr c Ethanol (% v/v) c ± a ± a ± c ± b ± ab ± 0.11 Total acidity (g/l) e ± b ± a ± d ± c ± b ± 0.24 Volatile acidity (g/l) 0.28 a ± c ± a ± a ± a ± b ± 0.02 Glucose (g/l) 0.03 a ± b ± a ± a ± a ± a ± 0.00 Fructose (g/l) 0.58 a ± b ± a ± a ± a ± a ± 0.09 Glycerol (g/l) 7.38 a ± c ± c ± a ± a ± b ± 0.18 a Data are representative of three independent experiments. Within each row, data followed by the same letter are not statistically significantly different (Tukey s honest significant difference, p 0.05). b Total acidity as tartaric acid (g/l) and volatile acidity as acetic acid (g/l). c See Table 1 for definitions.

6 Schizosaccharomyces japonicus in Wine Production 271 glycerol levels were similar to that of the control with S. cerevisiae in pure culture. Volatile organic compounds. Volatile organic compounds (VOCs) were analyzed across the wines to determine the potential impact of Sch. japonicus on VOCs in pure and mixed cultures compared to the control with only S. cerevisiae in pure culture (Table 4). A general increase in higher alcohols was observed in all the fermentations carried out by Sch. japonicus in pure culture, in free and immobilized form, as compared with the relevant control (SCF). Cell immobilization (SZI) resulted in a higher production of higher alcohols than those found in the trial with free cells (SZF). Significant increases of 2-methyl- 1-propanol (isobutanol), 3-methyl-1-butanol (isoamyl alcohol), and 1-hexanol compared with the relevant control (SCF), were observed. In contrast, a higher concentration of 1-propanol was present in the EC1118 control (SCF) than in all the trials with Sch. japonicus in pure cultures or mixed culture with S. cerevisiae. Sch. japonicus in pure culture in the free form or immobilized (SZF and SZI) displayed higher production of ethyl acetate, with levels being highest for immobilized cells reaching a quantity of ~0.46 g/l. The mixed fermentations showed intermediate concentrations of ethyl acetate, reflecting the additive contribution of Sch. japonicus cells in free or immobilized form. However, on the second day of the alcoholic fermentation, the concentration of ethyl acetate in the mixed fermentations with immobilized cells of Sch. japonicus (SZI + SCF and SZI + SCF 48 hr) was 81 and 44 mg/l, respectively (Supplemental Table 1). On the second day of the alcoholic fermentation, immobilized cells of Sch. japonicus in mixed fermentations with S. cerevisiae (SZI + SCF) produced high levels of some important aroma compounds, such as isoamyl acetate, hexyl acetate, phenethyl acetate, ethyl butyrate, and ethyl hexanoate, and at concentrations significantly higher than those produced by S. cerevisiae in single culture (SCF) (Supplemental Table 1). A significant increase in acetaldehyde was observed for the SZF + SCF trial (376 mg/l) compared with the relevant control with S. cerevisiae in pure culture (SCF) (27 mg/l) and the two pure culture samples of Sch. japonicus (67 and 134 mg/l in SZF and SZI, respectively). Free and immobilized cells of Sch. japonicus produced a significantly higher amount of acetoin (3-hydroxy-2-butanone) (667 mg/l and 555 mg/l, respectively) than the relevant control of Saccharomyces in Table 3 Malic acid (g/l) degradation by the yeasts during alcoholic fermentation. a Code b 3 days 6 days 14 days SCF 2.52 ± ± ± 0.01 SZF 1.35 ± ± ± 0.00 SZI 1.15 ± ± ± 0.01 SZF + SCF 2.50 ± ± ± 0.02 SZI + SCF 2.08 ± ± ± 0.03 SZI + SCF 48 hr 1.18 ± ± ± 0.03 a Data represent three independent experiments. b See Table 1 for code definitions. pure culture (10 mg/l). In mixed fermentations with S. cerevisiae, intermediate concentrations of acetoin (186 mg/l and 153 mg/l, in SZF + SCF and SZI + SCF 48 hr, respectively) were found, except for the trial SZI + SCF, which showed a concentration similar to that of the control with S. cerevisiae in pure culture. All pure and mixed-culture fermentations with Sch. japonicus had a concentration of the acetate esters (excluding ethyl acetate) two to three times higher than that present in the pure control fermentation with S. cerevisiae (SCF). In contrast, a decrease in ethyl ester compounds was observed in the trials carried out by Sch. japonicus in pure and mixed fermentations compared with the control with S. cerevisiae, except for ethyl butyrate, ethyl isobutyrate, and ethyl hexanoate. The concentration of ethyl isobutyrate was ~10-fold higher in the trials with pure culture of S. japonicus, in both free and immobilized form, compared to that with S. cerevisiae in pure culture. A significant increase in isobutyric acid and isovaleric acid concentrations was observed in the pure cultures of Sch. japonicus compared to S. cerevisiae in pure culture, and an increase was also observed in the mixed fermentations. Sch. japonicus showed a general decrease in medium fatty acids such as octanoic acid and decanoic acid, except for hexanoic acid, compared to the control with S. cerevisiae in pure culture. Polysaccharide release. The concentrations of polysaccharides released during the alcoholic fermentation by S. cerevisiae and Sch. japonicus in pure or mixed cultures are reported in Table 5. In pure culture, Sch. japonicus produced ~2.6- (day 3), 3.2- (day 6), and 4.7-fold (day 14) higher concentrations of polysaccharide than did S. cerevisiae, resulting in an average concentration of 840 mg/l (day 6) and 1041 mg/l (day 14) for Sch. japonicus (SZF). Cell immobilization (SZI) reduced polysaccharides release; however, at the end of the fermentation, SZI reached almost the same quantity released by S. japonicus in the free form, approximately five times higher than that released by S. cerevisiae (223 mg/l). In both mixed coinoculated fermentations (SZF + SCF and SZI + SCF), significant reductions of polysaccharide concentrations (~70% and 62%, respectively) were observed, compared with those obtained in the pure-culture fermentations with Sch. japonicus. However, a reduction of only 21% was observed in the mixed sequential fermentation trial (SZI + SCF 48 hr). Wine protein haze formation. Wine polysaccharide content has been associated with increased protein stability in wine (Dupin et al. 2000). Therefore, protein stability was assessed in all wines as turbidity after protein denaturation (Bentotest) or phenolic precipitation by interaction with reactive tannins (Proteotest), and measured as nephelometric turbidity units (NTUs) in a nephelometer (Figure 3). Samples were considered protein-unstable when the difference between treated and untreated samples was >10 NTUs or >15 NTUs, for Bentotest and Proteotest, respectively. Generally, the Bentotest produced higher turbidity values than the Proteotest. The Bentotest is known to precipitate all proteins in the sample, often resulting in an overestimate

7 272 Domizio et al. Table 4 Volatile compounds (mg/l) of wines after 14 days of fermentation. a Analyte ISTD b RT b SCF c SZF c SZI c SZF + SCF c SZI + SCF c SZI + SCF 48 hr c Higher alcohols 1-Propanol 1-Butanol d d ± a ± bc ± bc ± c ± b ± Butanol 1-Butanol d a ± b ± e ± b ± c ± d ± Methyl-1-propanol 1-Butanol d a ± c ± d ± a ± b ± d ± Methyl-1-butanol 5-Methyl-1-hexanol a ± c ± c ± b ± b ± c ± Hexanol 5-Methyl-1-hexanol a ± e ± d ± c ± b ± c ± Phenylethanol 3,4-Dimethyl phenol ab ± b ± b ± b ± b ± a ± 3.92 Total Oxigenated compounds Acetaldehyde Ethyl acetate d a ± ab ± c ± d ± ab ± bc ± hydroxy-2-butanone 1-Butanol d a ± d ± c ± b ± a ± b ± Total Acetate esters Ethyl acetate Ethyl acetate d a ± b ± e ± b ± c ± d ± 1.67 Isoamyl acetate Ethyl hexanoate d a ± c ± cd ± b ± d ± cd ± 1.05 Hexyl acetate Ethyl hexanoate d a ± bc ± cd ± b ± d ± bc ± Phenylethyl acetate Ethyl hexanoate d a ± c ± b ± b ± c ± c ± 0.34 Total Ethyl esters Ethyl butyrate Ethyl acetate d a ± b ± d ± b ± c ± c ± 0.03 Ethyl isobutyrate Ethyl acetate d a ± c ± d ± b ± a ± c ± 0.02 Ethyl hexanoate Ethyl hexanoate d a ± c ± ab ± c ± d ± b ± 0.11 Ethyl octanoate Ethyl hexanoate d c ± ab ± a ± b ± c ± a ± 0.00 Ethyl decanoate Ethyl hexanoate d d ± ab ± a ± bc ± cd ± a ± 0.04 Ethyl tetradecanoate Ethyl hexanoate d b ± a ± a ± b ± c ± a ± 0.00 Ethyl lactate 1-Butanol d c ± ab ± a ± c ± ab ± b ± 0.03 Diethyl succinate Ethyl hexanoate d c ± a ± a ± b ± c ± a ± 0.00 Total Fatty acids short-chain Isobutyric acid Hexanoic acid d a ± e ± cd ± d ± ab ± bc ± 0.25 Isovaleric acid Hexanoic acid d a ± e ± d ± c ± b ± d ± 0.05 Medium-chain Hexanoic acid Hexanoic acid d a ± b ± a ± bc ± c ± a ± 0.48 Octanoic acid Hexanoic acid d c ± ab ± a ± b ± c ± a ± 0.01 Decanoic acid Hexanoic acid d b ± b ± a ± b ± b ± a ± 0.01 Total a Data are representative of three independent experiments. Within each row, values followed by the same letters are not statistically significantly different (Tukey s honest significant difference, p 0.05). b ISTD: internal standard, RT: retention time. c See Table 1 for definitions.

8 Schizosaccharomyces japonicus in Wine Production 273 of the amount of bentonite needed (Dubourdieu et al. 1988). However, the turbidity values obtained here were considerably higher than the threshold value of protein stability of 10 NTUs recommended for defining a wine as protein-stable by this test. Wines obtained from single-culture fermentation of S. cerevisiae (SCF) and from the coinoculated free cells of S. cerevisiae/sch. japonicus (SCF + SZF) displayed the highest turbidity values. These results were consistent with the polysaccharide contents of the wines. Discussion In the last two decades, many non-saccharomyces yeasts have been proposed to be used in combination with Saccharomyces yeasts in winemaking to obtain wines with unique characteristics (Ciani et al. 2010). Non-Saccharomyces yeasts can affect wine flavor, aroma, and mouthfeel (Bisson et al. 2017). The yeast species Sch. japonicus had not been previously evaluated for use in winemaking, and our study has demonstrated several interesting enological features of this yeast when used during the alcoholic fermentation of Trebbiano grape juice. An important aspect to consider in mixed fermentations is the degree of competition between the yeast starters. In the present study, the growth and fermentation kinetics of the S. cerevisiae strain in pure culture (SCF) were comparable to Table 5 Polysaccharides (mg/l) released by the yeasts during alcoholic fermentation. a Code b 3 days 6 days 14 days SCF ± ± ± SZF ± ± ± SZI ± ± ± SZF + SCF ± ± ± 7.32 SZI + SCF ± ± ± SZI + SCF 48 hr ± ± ± a Data represent three independent experiments. b See Table 1 for code definitions. Figure 3 Wine turbidity values (as NTUs), measured with a nephelometer after the addition of phosphomolybdic acid (Bentotest) or reactive tannins (Proteotest). Data represent three independent experiments. Error bars show standard deviations of three independent experiments. See Table 1 for definitions of x-coordinates. those observed in the coinoculated fermentations with free and immobilized cells of the UCD2489 strain Sch. japonicus (SZF + SCF and SZI + SCF). In contrast, a lower fermentation rate was observed in the sequential fermentation (SZI + SCF 48 hr). This may have been due to the low fermentative activity of Sch. japonicus, but also may have been caused by the slower growth of S. cerevisiae during the first four days in the sequential fermentation. However, the fermentations did not arrest in any case. Thus, depending on inoculation protocols, use of Sch. japonicus had little impact on completion of the fermentation by Saccharomyces. It is worth noting that Sch. japonicus cells flocculated after the second day of alcoholic fermentation. Flocculation in fission yeasts has been shown to represent an initial and essential step in the process of sexual conjugation and subsequent sporulation (Calleja and Johnson 1971, Calleja et al. 1977). Sch. japonicus cells usually enter meiosis following a deterioration in nutrient conditions (Niki 2014). However, in the present study, the early onset of the conjugation process appeared not to be linked to a nitrogen deficiency, but rather to other specific stress conditions. Thus, flocculation in this case may have been due to the presence of other environmental conditions, either leading to passive changes in cellsurface structures due to differences in bonding capacities of cell-surface components, or reflecting an active adaptation to growth under these conditions with deliberate release and replacement of cell-surface components. The accompanying change of cell-surface composition could then enhance flocculation for reasons not associated with sexual reproduction. Other explanations are also possible. Sch. japonicus cells consumed lower amounts of ammonia and primary amino nitrogen than those consumed by the pure culture of S. cerevisiae, suggesting that the Sch. japonicus cells were arresting growth and metabolism because of the development of nonpermissive conditions within the fermentation. Benito et al. (2012, 2016a, 2016b) also observed a reduced nitrogen demand in alcoholic fermentations with Sch. pombe compared with those carried out by S. cerevisiae. There is a greater nitrogen release and higher final concentrations of most amino acids with Sch. pombe. Similarly, Sch. japonicus might affect nitrogen dynamics of mixed- and pureculture fermentations, but this was not evaluated in our study. Alternatively, the slower growth of S. cerevisiae could be related to the growth of Sch. japonicus, permitting release of a potential inhibitory factor or depletion of nutrients. Taillandier et al. (1995) reported that Saccharomyces growth is inhibited by Sch. pombe and that this inhibition is proportional to Sch. pombe cell concentration. The inhibition was proposed as an amensal effect similar to production of a killer factor; however, these authors did not isolate the killer protein or confirm its existence. The slower growth and fermentation kinetics of S. cerevisiae in the sequential fermentation (SZI + SCF 48 hr) could not be solely explained by nitrogen depletion by Sch. japonicus during the first days of the alcoholic fermentation. After three days of fermentation, the pure cultures of Sch. japonicus (both in immobilized and free form) used less

9 274 Domizio et al. ammonia and primary amino nitrogen than did the pure culture of S. cerevisiae. Moreover, at the end of the fermentation of all mixed cultures, an increase in primary amino nitrogen was observed, dictated mainly by the behavior of S. cerevisiae and probably also arising from yeast autolysis (Andorrà et al. 2010). Further investigations are necessary to clarify the specific growth needs and stress sensitivities of the Sch. japonicus species. In the coinoculated fermentations with non-immobilized Sch. japonicus (SZF + SCF), the higher S. cerevisiae-to-sch. japonicus inoculum ratio of 10:1 than the 1:1 ratio used in the cultures with immobilized Sch. japonicus cells (SZI + SCF) likely caused S. cerevisiae to dominate from the beginning of the alcoholic fermentation and to give a higher final ethanol concentration. The lower alcohol yield in the mixed fermentation with immobilized Sch. japonicus and S. cerevisiae may represent an advantage in fermentations of musts with high initial sugar content, allowing wines with a lower alcohol content to be obtained. Reducing wine alcohol content, to meet the growing international consumer demand for low-alcohol wines, is currently an important objective for various wine producers (Ciani et al. 2016). In this context, it is worth considering that cell encapsulation in alginate creates a cell-protective layer that makes the cells resistant to ethanol (Norton et al. 1995), thereby improving fermentation performance with respect to free cells. The presence of this protective matrix could be also the reason for the higher volatile acidity in the Sch. japonicus pure culture inoculated with free, rather than immobilized, cells. Moreover, as stated above, the dominance of S. cerevisiae in the coinoculated trials with free cells of S. japonicus (SZF + SCF) likely resulted in the minor influence of Sch. japonicus on the alcoholic fermentation and, in turn, in lower production of acetic acid. The production of high acetic acid levels represents one of the major problems observed with the use of Sch. pombe yeasts during the alcoholic fermentation of grape juice. However, a high variability in volatile acidity has been observed for these yeasts. Benito et al. (2016b), evaluating 75 Sch. pombe strains, found that most of the strains (65%) produced acetic acid ranging from 0.4 g/l to 0.7 g/l, 15% of the strains between 0.8 and 1.4 g/l, and only 8% of the strains produced less than 0.4 g/l acetic acid. Instead, Du Plessis et al. (2017) reported final levels of volatile acidity below 0.1 g/l for one Sch. pombe strain, up to five times lower than those produced by several S. cerevisiae controls. These results suggest that a selection process similar to that performed by Benito et al. (2016b) for Sch. pombe could be used to select Sch. japonicus strains that are more appropriate for winemaking. Malic acid degradation was influenced by the cell ratio of Sch. japonicus to S. cerevisiae and appeared to be adversely affected by S. cerevisiae when present from the beginning of the alcoholic fermentation. In the coinoculated fermentations (SZF + SCF and SZI + SCF), less malic acid was metabolized than in the sequential fermentation (SZI + SCF 48 hr). Partial consumption of malic acid has not been observed for Sch. pombe (Taillandier et al. 1995, Benito et al. 2016b) because this yeast species reaches sufficient concentrations to permit total malic acid depletion. These results are particularly interesting because they indicate some measure of control over the extent of deacidification by manipulation of the Sch. japonicus inoculum cell concentration. If a subsequent malolactic fermentation is desired, using a strain that does not deplete malate levels may be preferable to strains that degrade the available malate. Another interesting Sch. japonicus feature was the production of glycerol at levels ~2-fold higher than those released by S. cerevisiae. In contrast, Benito et al. (2016b) did not find significant differences in glycerol production among six different strains of Sch. pombe, and between these strains and two different S. cerevisiae strains. Glycerol is thought to be an important compound affecting sweetness of wine (Noble and Bursick 1984). Therefore, additional studies are necessary to confirm this phenotypic trait among different strains of Sch. japonicus and to further analyze their behavior during the winemaking process. Regarding the production of volatile compounds by this non-saccharomyces yeast, a higher quantity of higher alcohols was present in the trials with immobilized Sch. japonicus cells (SZI) than in those with free cells (SZF). The influence of cell immobilization on bioflavor formation has been explored in wine, beer, and other alcoholic beverages (Nedović et al. 2015). In particular, higher alcohols have been reported to act as quorum-sensing molecules produced by yeasts to adapt to their environment in response to population density. As a consequence, high cell densities together with high concentration of specific higher alcohols can potentially promote production of precursors of higher alcohols and esters (Nedović et al. 2015). However, in both types of mixed fermentations, an increase in higher alcohol occurred likely because of Sch. japonicus metabolism. Higher production of ethyl acetate was observed in the trials with immobilized cells of Sch. japonicus (SZI), compared to that found in the trials with free cells (SZF). In this context, Shen et al. (2003) reported changes induced by cell immobilization in the expression levels of those genes encoding enzymes involved in acetate ester formation, such as alcohol acetyltransferases. Intermediate concentrations of ethyl acetate were present in the mixed fermentations and reflected the additive contribution of Sch. japonicus cells. In all trials, ethyl acetate was far above the reported detection level of 150 mg/l in wine (Lambrechts and Pretorius 2000), except for the trial with Sch. japonicus in coinculation with S. cerevisiae (SZF + SCF) ( mg/l). This decrease in ethyl acetate formation may have been due to the aforementioned greater dominance of S. cerevisiae in these fermentations. Ethyl acetate is associated with a solvent -like aroma (Swiegers et al. 2016), and is considered detrimental for wine quality when present at detectable concentrations. However, within the second day of the alcoholic fermentation, in the mixed fermentations with immobilized Sch. japonicus cells (SZI + SCF and SZI + SCF 48 hr), the ethyl acetate concentration was below its detection level, and some important aroma compounds accumulated to higher levels than those produced

10 Schizosaccharomyces japonicus in Wine Production 275 by S. cerevisiae in single culture (SCF). These results suggested that removal of the immobilized Sch. japonicus cells within the second or third day of alcoholic fermentation could help manage ethyl acetate production and, at the same time, could take advantage of the production of interesting volatile compounds. Acetaldehyde production exhibited trends similar to those for ethyl acetate. The increase in acetaldehyde could be explained by synergistic interactions between the two yeasts, both present in the free form, and not by an additive effect. Possible cell-to-cell contact signaling could be the reason for these interactions (Richard et al. 1996). Moreover, higher acetaldehyde production has been reported as a frequent side effect of yeast immobilization (Nedović et al. 2015). As is the case for ethyl acetate, acetaldehyde is normally associated with off-flavors ( green, grassy, or apple-like ) when present in wine at high concentrations (Swiegers et al. 2016). However, the amount of acetaldehyde in both mixed fermentations with Sch. japonicus in the immobilized form was lower than the reported threshold value of 100 mg/l in wine (Lambrechts and Pretorius 2000). Benito et al. (2016b), evaluating the production of volatile compounds by six different Sch. pombe strains, found that two strains in particular yielded higher concentrations of acetaldehyde and ethyl acetate, ranging between 32 and 36 mg/l, and 82 and 89 mg/l, respectively. Morata et al. (2012) also reported high production of acetaldehyde by Sch. pombe. This compound, together with pyruvic acid, has been associated also with red wine color stabilization (Benito et al. 2017). Indeed, various studies have shown that acetaldehyde mediates anthocyanin-tannin and tannin-tannin condensation reactions, permitting generation of stable pigments in wine (Bakker and Timberlake 1997, Eglinton et al. 2004). Further studies are necessary to assess the possible influence of acetaldehyde produced by Sch. japonicus on wine color stability during the vinification of red grape juice and to investigate residual concentrations of free acetaldehyde in the final wine resulting from these reactions. Microbial production of acetaldehyde early in fermentation may enable chemical reactions to occur earlier than those normally happening during aging in the presence of oxygen and oxidative generation of acetaldehyde. This last compound might react with the anthocyanins to produce derivatives such as vitisin B (malvidin- 3-O-glucoside-4-vinyl), a compound that also provides stable color to aged wine (Morata et al. 2012). The higher amounts of acetoin in the mixed fermentations SZF + SCF and SZI + SCF 48 hr than in the relevant control of Saccharomyces in pure culture, reflect the additive contributions of Sch. japonicus cells. The low production of acetoin by Saccharomyces was in agreement with previously reported findings (Romano and Suzzi 1996). The low amounts of acetoin in in mixed fermentation SZI + SCF may have been due to its use by S. cerevisiae to produce 2,3-butanediol, as acetoin is a key compound in the biosynthesis of 2,3-butanediol and diacetyl. It is worth noting that acetoin has a relatively high odor threshold (150 mg/l), and its impact on wine aroma therefore is almost always irrelevant. Among the other volatile compounds, a significant increase in acetate esters was observed in the fermentation carried out by pure culture of Sch. japonicus, in both free and immobilized form, compared to the control with S. cerevisiae (SCF). Acetate esters are responsible for fruity and floral aroma. In this regard, compounds such as isoamyl acetate (fruity, such as banana and apple), hexyl acetate (fruity, such as pear, floral), and phenyl ethyl acetate (floral, such as roses), have low threshold values (0.03 mg/l, 0.7 mg/l, and 0.25 mg/l, respectively) (Swiegers et al. 2016). As a result, higher amounts of these compounds in the mixed fermentation, relative to the control with S. cerevisiae only, may have played an important role in the final sensory characteristic of the relevant wines. Despite the lower concentrations of most ethyl ester compounds in the trials carried out by Sch. japonicus in pure and mixed fermentations than in the control with S. cerevisiae, ethyl butyrate and ethyl isobutyrate were present at significantly higher concentrations in the sequential fermentation (SZI + SCF 48 hr), reflecting the additive contributions of Sch. japonicus cells in immobilized form, and likely highlighting the influence of the cell-immobilization technique on the production of these compounds. The production of isobutyric, isovaleric, and hexanoic acids was significantly higher in the immobilized form of the pure culture of Sch. japonicus (SZI) than in the free cells, highlighting a possible influence of the immobilization technique on cell-membrane composition. Indeed, Norton and D Amore (1994) suggested that cell immobilization can modify fatty acid concentration in cell membranes because of oxygen diffusion limitations. Medium fatty acids have been recognized as having toxic effects toward yeasts (Swiegers et al. 2016). The lower concentration of these compounds in both pure and mixed culture with Sch. japonicus, compared to the control with S. cerevisiae in pure culture, suggests that the possible negative effect on S. cerevisiae growth observed in Figure 1 was not due to the production of these compounds, but this requires additional evaluation. Further studies are also necessary to evaluate the production of sulfur compounds by Sch. japonicus during the alcoholic fermentation. Indeed, the production of H 2 S has been reported to be one of the major drawbacks of fermentations with Schizosaccharomyces yeasts (Yokotsuka et al. 1993). The SPME method employed in the present study was developed at 60 C to sample either high- or low-boiling compounds (such as decanoic acid, C12 14 esters, and acetaldehyde), and, as a consequence, it was not optimized to retrieve low-boiling sulfur compounds such as H 2 S, methanethiol, or ethanethiol at trace concentration. However, it is worth noting that no H 2 S production was observed by plating the strain Sch. japonicus UCD2489 onto Biggy Agar (data not shown). Moreover, no off-characters related to sulfur VOCs were detected by an informal sensory evaluation of the wines. Particularly interesting was the high quantity of polysaccharides released by Sch. japonicus into the media, confirming in actual grape juice what has previously been observed in synthetic grape juice fermentations (Domizio et al. 2017). However, a significant reduction in polysaccharides