RESEARCH ARTICLE

The influence of Wickerhamomyces anomalus killer yeast on the fermentation and chemical composition of apple wines Pawel Satora, Tomasz Tarko, Pawel Sroka & Urszula Blaszczyk Department of Fermentation Technology and Technical Microbiology, University of Agriculture, Krakow, Poland

Correspondence: Pawel Satora, Department of Fermentation Technology and Technical Microbiology, University of Agriculture, ul. Balicka 122, Krakow, Poland. Tel.: +48 12 662 47 97; fax: +48 12 662 47 98; e-mail: [email protected] Received 15 November 2013; revised 28 March 2014; accepted 15 April 2014. Final version published online 8 May 2014. DOI: 10.1111/1567-1364.12159 Editor: Isak Pretorius Keywords Wickerhamomyces anomalus; mixed cultures; spontaneous fermentation; killer yeast; volatile compounds; GC-SPME.

Abstract The aim of this study was to determine the influence of two different Wickerhamomyces anomalus strains, CBS 1982 and CBS 5759, on the chemical composition and sensory characteristics of Gloster apple wines. They were inoculated into unpasteurized as well as pasteurized apple musts together with a S. cerevisiae strain as a mixed culture. Fermentation kinetics, basic enological parameters, antioxidant properties as well as selected polyphenol, volatile compound, and organic acid contents were analyzed during the experiments. Apple wines obtained after spontaneous fermentation were characterized by high volatile acidity, increased concentrations of acetaldehyde, and volatile esters, as well as the lowest amounts of ethyl alcohol and higher alcohols compared with other samples. Addition of 0.05 g L 1 W. anomalus killer strains to the unpasteurized must significantly changed the fermentation kinetics and chemical composition of apple wines. The value of volatile acidity was highly decreased, while the amount of higher alcohols and titratable acidity increased. Pasteurization of must improved the fermentation efficiency. Higher amounts of polyphenol compounds and lower amounts of malic acid were also detected. Application of W. anomalus strains together with S. cerevisiae yeast as a mixed culture positively influenced the chemical composition and sensory features of produced apple wines.

YEAST RESEARCH

Introduction Wickerhamomyces anomalus (formerly Pichia anomala) is an ascomycetous heterothallic yeast of the family Wickerhamomycetaceae that reproduces asexually by budding and sexually by the formation of hat-shaped ascospores (Kurtzman & Fell, 1998). Strains of this species are present in many types of environments and have been isolated from fruit and plant materials, cereal grain, maize silage, highsugar food products, and wine (Kurtzman & Fell, 1998). Wickerhamomyces anomalus is classified as a biosafety level 1 organism that is considered safe for healthy individuals. Wickerhamomyces anomalus can grow under extreme environmental stress conditions, such as low and high pH, low water activity, high osmotic pressure and anaerobic conditions. Due to these characteristics, this yeast can be a spoilage organism, for instance in high-sugar food products. Although yeast able to grow over a broad pH range and at high osmotic pressure, W. anomalus is not particularly tolerant to ethanol and acetate (Passoth et al., 2006). FEMS Yeast Res 14 (2014) 729–740

During wine fermentation, Wickerhamomyces strains predominate in the middle stages when ethanol levels reach 3–4% (Rojas et al., 2003). Wickerhamomyces anomalus is one of the stronger producers of isoamyl acetate (associated with descriptors such as banana) as well as ethyl acetate and acetic acid in pure culture, leading to serious wine deterioration (Suarez-Lepe & Morata, 2012). Occasionally, it can be tolerant to molecular sulfite levels (Warth, 1985). There is also a considerable amount of published information on the widely intergeneric killing spectrum of Wickerhamomyces toxins. Among the species with a killer phenotype, W. anomalus NCYC 434 has been extensively studied, and its killer protein, panomycocin, has been suggested as a potential antifungal agent (Platania et al., 2012). Panomycocin is a glycosylated monomeric protein with a molecular mass of 49 kDa. It belongs to the exo b-1,3 glucanase group and exerts its cytocidal activity by hydrolyzing the b-1,3-glucans, which are the primary _ u et al., 2005). polymers within the fungal cell wall (Izg€ Killer toxins from W. anomalus have been investigated as ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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antimicrobial agents against the spoilage yeast Dekkera/ Brettanomyces (Comitini et al., 2004). This has a potential application during wine maturation and storage. Other Pichia spp., that is, P. anomala, P. membranifaciens and P. subpelliculosa, together with Williopsis saturnus, have also been suggested for the production of low-alcohol wines (c. 3% v/v) in an aerated vessel (Erten & Campbell, 2001). The aim of this study was to determine the influence of two different W. anomalus strains on the chemical composition and sensory characteristics of apple wines. They were inoculated into unpasteurized as well as pasteurized apple musts together with S. cerevisiae strain as a mixed culture; thus, their influence on the natural microbiota and modification of apple wine composition was evaluated. Gloster apples, chosen for the research, are one of the most popular varieties of dessert apples grown in Poland.

Materials and methods Yeast, plant material, and fermentation

Active wine yeast Saccharomyces cerevisiae cv. Johannisberg-Riesling (JR; Culture Collection of the Fermentation Technology and Technical Microbiology Department of Agricultural University of Krakow) and killer yeast of Wickerhamomyces anomalus (CBS 1982, K4 and CBS 5759, K8) were used for the fermentation. Apple musts were obtained from Gloster apples (harvested in October 2009 from the experimental apple orchard in Garlica Murowana near Krakow) by treatment of crushed fruits with the pectinolytic preparation Pektopol PT-400 (0.3 mL kg 1; Pektowin, Jasło, Poland) for 3 h at 28 °C and pressing the fruit pulp. Sucrose (up to 22°Bx) was then added. Part of the must was pasteurized for 10 min at 95 °C, and the other part was unpasteurized. Alcoholic fermentation was conducted for 28 days at 25 °C in 3-liter glass flasks containing 2 L of apple must. Some samples were fermented spontaneously, while others were inoculated with S. cerevisiae (0.4 g dry weight per liter) and/or W. anomalus (0.04 g DW L 1). The weight losses associated with the liberation of carbon dioxide were measured daily. After fermentation, the young wine was separated from the sediments by carefully pouring into another vessel and kept for further clarification (sedimentation under gravity) for 48 h at 4 °C. Clarified young wines were subjected to analysis. All samples were tested in triplicate. Enological parameters analysis

The ethanol content, volatile acidity, total extract, sugarfree extract, reducing sugars, and sucrose concentrations were determined using official methods (OIV, 2005). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

pH and titratable acidity (TA) were determined using a Titrator Mettler DL 25 equipped with a printer (Switzerland). Titratable acidity was calculated from the volume of NaOH used for titration and expressed as g L 1 of malic acid. Total antioxidant activity assay (TAA)

Total antioxidant activity was determined using the ABTS method (Miliauskas et al., 2004). ABTS˙+ radical was generated by oxidation of ABTS with potassium persulfate K2S2O8 (POCh SA, Gliwice, Poland). Once the radical was formed, 0.1 mL of diluted wine sample was added to 1 mL of ABTS˙+ radical cation and the absorbance was measured at 734 nm with a spectrophotometer (DU-650, Beckman Instruments, Inc., Fullerton). Standard Trolox solutions (40–200 lM) were also evaluated against the radical to obtain a calibration curve. Results were expressed as milligrams of Trolox equivalent (TE) per 100 mL of sample. Total phenol index (TPI)

The amount of total phenols in wines was determined according to the Folin–Ciocalteu colorimetric method (Waterhouse, 2002). Wine samples were diluted with water (1 : 4). A 1 mL volume of the standard or sample solution was added to 5 mL of Folin–Ciocalteu reagent (1 : 10 dilution; Sigma-Aldrich), 50 mL of deionized water and 20 mL of sodium carbonate (20% w/v). The reaction mixture was then made up to the mark in a 100mL volumetric flask and was left to stand for 30 min before measuring the absorbance at 765 nm (Beckman DU-650 spectrophotometer). A calibration curve was obtained with gallic acid solutions (concentration range 0.4–5 mg L 1; Fluka), and the results are expressed as milligrams of gallic acid per liter of wine. Polyphenols analysis (HPLC)

HPLC apparatus consisting of a Merck-Hitachi L-7455 diode array detector and quaternary pump L-7100 equipped with the D-7000 HSM multisolvent delivery system (Merck-Hitachi, Tokyo, Japan) was employed. Separation was performed on a Synergi Fusion RP-80A 150 9 4.6 mm (4 lm) Phenomenex (Torrance, CA) column thermostated at 30 °C. The mobile phase was composed of solvent A (2.5% acetic acid) and solvent B (acetonitrile). The program began with a linear gradient from 0% B to 36 min 25% B, followed by washing and reconditioning the column. The flow rate was 1 mL min 1, and the runs were monitored at the following wavelengths: flavonols at 280 nm, phenolic acids at 320 nm, flavonols at FEMS Yeast Res 14 (2014) 729–740

Influence of W. anomalus yeast on apple wines composition

360 nm and anthocyanidins at 520 nm. Retention times and spectra were compared to those of pure standards within 200–600 nm. In addition, enzymatic hydrolysis of flavonol glycosides in citrate buffer solution (pH 5.0) was performed using the following enzymes: b-glucosidase, b-xylosidase, b-galactosidase, and b-hesperidinase (Sigma, Steinheim, Germany). The disappearance of single peaks in the chromatogram and formation of the corresponding aglycone was observed using HPLC after 1-h incubation at 38 °C with a specific enzyme. Results (expressed as mg L 1 of apple wine) were read from standard curves developed for the corresponding standards: chlorogenic acid, caffeic acid, p-coumaric acid, p-coumaroylquinic acid, (+) catechin, (-) epicatechin, phloridzin, and quercetin glycoside manufactured by the Sigma-Aldrich company. To determine the magnitude of error in the selected series, the assays were repeated. The standard error in the HPLC assays was below 10%. Organic acids analysis (HPLC)

The wines were centrifuged (15 min, 2154 g, 20 °C) and diluted fivefold using deionized water after filtering through a 0.45-lm membrane filter. A Perkin Elmer Flexar HPLC system equipped with a pump system and a UV/Vis detector monitored at 210 nm were used for the analysis. Tartaric, malic, acetic, lactic, citric, and succinic acids (Sigma-Aldrich) were analyzed on a LiChrosorb RP18 column (10 m, 25 cm 9 4.0 mm). The samples were eluted isocratically at 40 °C with mobile phase (0.045N H2SO4) at flow rate 0.4 mL min 1.

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column was heated using the following temperature program: 35 °C for 5 min at an increment of 5 °C per min to 110 °C, then 40 °C per min to 220 °C and maintaining a constant temperature for 3 min. The carrier gas was helium at a 20.0 mL min 1 flow. Hydrogen flow speed was 33.0 mL min 1, and that of air was 400 mL min 1. The qualitative and quantitative identification of volatile substances (acetaldehyde, acetone, ethyl acetate, isoamyl acetate, ethyl capronate, methanol, propanol, isobutanol, amyl alcohols, and 2-phenylethanol; SigmaAldrich) was based on the comparison of retention times and peak surface area read from sample and standard chromatograms. All tests were carried out in triplicate. Sensory analysis

Sensory assessment of apple wine samples was performed using the Buxbaum model of positive ranking (Satora & Tuszy nski, 2010). This model is based on 4 sensorial experiences rated by a maximum of 20 points. The samples of apple wines were subjected to sensory evaluation by a panel comprising 10 qualified testers, all of them highly experienced in sensory testing. Statistical analysis

19.0 software was applied for statistical results analyses. Statistically significant differences between results (P = 0.05) were evaluated using one-way analysis of variance (ANOVA).

SPSS

Results

Volatile compounds analysis (GC-SPME)

Enological characteristics of apple wines

Two milliliters of each wine sample was transferred to a 15 mL amber vial having screw caps (Supelco) with a magnetic stirrer and 1 g of NaCl, which was then spiked with 2 lL of internal standard (4-methyl-2-pentanol; Fluka). The SPME device (Supelco Inc., Bellefonte, PA) coated with PDMS (100 lm) fiber was first conditioned by inserting it into the GC injector port at 250 °C for 1 h. For sampling, the fiber was inserted into the headspace under magnetic stirring (300 r.p.m.) for 35 min at 40 °C. Subsequently, the SPME device was placed in the injector port for chromatographic analysis and was left in the inlet for 2 min. The GC-SPME analysis was performed on a Hewlett Packard 5890 Series II chromatograph system. The tested components were separated on a HP-INNOVAX capillary column (cross-linked polyethylene glycol stationary phase; 30 m 9 0.53 mm ID with 1.0 lm film thickness). The detector and injector temperature was 250 °C, and the

The killer strains of Wickerhamomyces anomalus were added to uninoculated and unpasteurized must, as well as must inoculated with S. cerevisiae wine strain. The kinetics of these fermentations are shown in Fig. 1. The turbulent stage of spontaneous fermentation appeared after 24 h of fermentation. Addition of W. anomalus CBS5759, producer of K8 killer toxin, to an unpasteurized must, speeded up the fermentation and resulted in the highest final weight losses due to the liberation of carbon dioxide. The opposite phenomenon was found during fermentation of musts with W. anomalus CBS1982 (K4 toxin producer). From the 8th day, a decrease in the rate of carbon dioxide evolution was observed. Killer yeasts are frequently used to combat and prevent contamination by wild-type yeasts during wine production, and they can even dominate the wine fermentation. If there is an unbalanced ratio of killer to sensitive yeasts, stuck and sluggish fermentations may result (Maturano et al., 2012).

FEMS Yeast Res 14 (2014) 729–740

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CO2evolved [g per 100 mL]

6.00 5.00 4.00

Spontaneous

3.00

Spont+K4 (nonpasteurized)

2.00

Spont+K8 (nonpasteurized)

1.00 0.00 0

2

5

7

9 11 13 15 17 19 21

Days CO2evolved [g per 100 mL]

6.00 5.00 4.00

S. cerevisiae (nonpasteurized)

3.00

JR+K4 (nonpasteurized)

2.00

JR+K8 (nonpasteurized)

1.00 0.00 0

2

5

7

9 11 13 15 17 19 21

Days CO2evolved [g per 100 mL]

6.00 S. cerevisiae (pasteurized)

5.00 4.00

JR+K4 (pasteurized)

3.00 2.00

JR+K8 (pasteurized)

1.00 0.00 0

2

5

7

9 11 13 15 17 19 21

Days Fig. 1. The fermentation kinetics of Gloster apple musts using spontaneous fermentation as well as Saccharomyces cerevisiae cv. Johannisberg-Riesling (JR) and/or Wickerhamomyces anomalus CBS 1982 (K8) and CBS 5759 (K4) yeast strains (standard deviations for all data were < 1%).

The killer strains used did not significantly influence the fermentation kinetics of S. cerevisiae in unpasteurized musts. Between the 1st and 17th day of fermentation, higher fermentation rate was found in pasteurized musts fermenting by mixed cultures of S. cerevisiae and W. anomalus, compared to musts fermenting by the pure S. cerevisiae strain. The resulting apple wines were characterized by varied amounts of residual sugars (Table 1). According to EU regulation 753/2002, most of them may be classified as dry (up to 4 g L 1 of sugars), and others as semi-dry wines (4–12 g L 1). The highest degree of fermentation was observed in wines fermented spontaneously with the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

addition of killer yeasts. JR+K8 (unpasteurized), JR+K4 (pasteurized), and JR+K8 (pasteurized) were distinguished by higher reducing sugar (and ethanol) concentrations compared with wines fermented by a monoculture of S. cerevisiae. In all samples, sucrose was utilized completely and the residual sugars constituted reducing sugars. Statistically significant differences were also found in the case of sugar-free extract concentration (Table 1). Its level depends mainly on glycerol amount and nonvolatile organic acids such as succinic acid (Ciani & Ferraro, 1998). In this respect, samples obtained by spontaneous fermentation were distinguished by over two times higher sugar-free extract level compared to that of inoculated fermentations. The sugar-free extract concentration was also determined by the yeast used for fermentation. Wickerhamomyces anomalus CBS5759 and S. cerevisiae strains generally reduced the sugar-free extract level, while W. anomalus CBS1982 increased it. Like aforementioned parameters, ethyl alcohol concentration of wines also showed significant variation (Table 1). The lowest amounts of ethanol were formed in samples fermented spontaneously (84–87 g L 1). Among wines produced using S. cerevisiae strain, those prepared from pasteurized must were characterized by the highest ethanol concentration. Both strains of W. anomalus caused an increase in ethanol level. The highest amount of ethanol was determined in samples after fermentation of pasteurized must with the mixed culture of S. cerevisiae and W. anomalus CBS1982 (102.6 g L 1). It was over 10% more compared to other inoculated samples and over 20% more compared to wines obtained after spontaneous fermentation. Titratable acidity was another analyzed enological parameter that was statistically significantly different among apple wines. Fresh apple musts were characterized by titratable acidity of 4.72 g L 1 expressed as malic acid. The wines produced contained less organic acids (Table 1). The lowest deacidification occurred during spontaneous fermentation of the musts. Inoculated fermentation contributed to a higher decrease in acidity, of about 1 g L 1. Pasteurization of must caused higher reduction in titratable acidity of produced wines (even up to 1.5 g L 1 in relation to unfermented must). At the same time, samples fermented by mixed cultures of W. anomalus and S. cerevisiae were distinguished by lower acidity compared with wines obtained using a monoculture of S. cerevisiae. Organic acids composition of apple wines

The analysis of selected organic acids (Table 2) in apple wines showed that the type of fermentation applied, FEMS Yeast Res 14 (2014) 729–740

Influence of W. anomalus yeast on apple wines composition

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Table 1. The main enological parameters of fresh apple must and apple wines obtained using spontaneous fermentation as well as Saccharomyces cerevisiae cv. Johannisberg-Riesling (JR) and/or Wickerhamomyces anomalus CBS 1982 (K8) and CBS 5759 (K4) yeast strains

Apple wines Fresh apple must Spontaneous Spontaneous+K4 Spontaneous+K8 JR JR JR+K4 JR+K4 JR+K8 JR+K8 Sig.

Pasteurization

Extract (g L 1)

Total sugars (g L 1)

+

115.0 33.3d (2.9) 23.3c (1.4) 21.7bc (1.4) 15.0a (0.0) 20.0b (1.5) 20.0b (0.0) 22.5c (0.0) 20.0b (0.0) 20.0b (0.0) ***

98.3 6.9fg (2.9) 0.0a 0.6ab (0.2) 3.3cd (0.3) 2.1bc (0.3) 2.2bc (0.2) 4.7de (0.4) 6.4ef (0.6) 8.4g (0.4) ***

+ +

Reducing sugars (g L 1)

Sucrose (g L 1)

Sugar-free extract (g L 1)

Ethanol concentration (g L 1)

Titratable acidity† (g L 1)

75.6 6.9fg (2.9) 0.0a 0.6ab (0.2) 3.3cd (0.3) 2.1bc (0.3) 2.2bc (0.2) 4.7de (0.4) 6.4ef (0.6) 8.4g (0.4) ***

21.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 –

17.8 26.4f (1.5) 23.3e (1.4) 21.1d (1.6) 11.7a (0.3) 17.9c (1.2) 17.8c (0.2) 17.8c (0.4) 13.6b (0.6) 11.6a (0.4) ***

0.0 84.4a (1.1) 83.9a (0.0) 87.2b (1.6) 88.0b (0.1) 94.7d (0.8) 91.9c (0.8) 102.6f (0.0) 94.9d (0.0) 97.6e (0.0) ***

4.72 3.95d (0.12) 4.47e (0.04) 4.51e (0.15) 3.32ab (0.00) 3.41b (0.28) 3.75c (0.00) 3.23ab (0.02) 3.71cd (0.04) 3.15a (0.12) ***

Values with different superscript roman letters (a-g) in the same column are significantly different according to the Duncan’s test (P < 0.05). † Expressed as g L 1 of malic acid. Sig., significance; *, **, *** display the significance at 5%, 1%, and 0.5% by least significant difference; ns, not significant.

pasteurization of must, as well as yeast strains used did not have a statistically significant effect on the concentration of tartaric, acetic, lactic and succinic acids. The amount of these components was 0.2–0.4, 1.0–1.4, 0.4–0.5 and 0.2–0.3 g L 1, respectively. However, a statistically significant difference in malic and citric acid concentrations was found. The amount of malic acid depended mainly on pasteurization of must and spontaneous fermentation. All samples prepared from pasteurized must contained up to 50% less malic acid compared to those produced without thermal treatment (Table 2). Assimilation of malic acid also increased in the case of mixed cultures of S. cerevisiae and W. anomalus. In these wines, the lowest concentrations of this component – 1.04 and 1.07 g L 1, respectively – were observed. Fermentation of unpasteurized must by epiphytic microbiota of apples also caused a significant reduction in malic acid. This was not found in samples which were spontaneously fermented with the addition of W. anomalus strain. Apple wines obtained after inoculation with S. cerevisiae and/or one of the strains of W. anomalus were characterized by the same level of citric acid ranging from 1.0 to 1.3 g L 1. Only the wine produced by spontaneous fermentation was distinguished from others by higher concentration of this compound, that is, 2.4 g L 1. Contrary to acetic acid amount measured by HPLC, the volatile acidity determined by titration was characterized by statistically significant differences (Table 2). The highest volatile acidity level was found in the wines after spontaneous fermentation; however, inoculation of unpasteurized must only with W. anomalus yeasts reduced its level by up to 50%. Pasteurization of musts also positively affected the amount of volatile acids (decrease by over 50%). FEMS Yeast Res 14 (2014) 729–740

Inoculation with mixed cultures of S. cerevisiae and W. anomalus generally did not influence the volatile acidity of apple wines with the exception of JR+K8 wines produced from unpasteurized must where a higher level of volatile acidity (0.81 g L 1) was observed. Antioxidant activity and polyphenolic composition of apple wines

Type of fermentation, pasteurization of must, and addition of W. anomalus yeast statistically significantly influenced the antioxidant activity as well as the content of selected polyphenols in apple wines (Table 3). Pasteurization of must generally increased the antioxidant capacity of samples measured using ABTS radical. The JR and JR+K4 wines were distinguished by up to 20 mg TE/100 mL higher activity compared with those prepared from unpasteurized must. The highest total polyphenol content was found in samples fermented spontaneously (324 mg TE/100 mL). This parameter was also higher in wines produced by mixed cultures of S. cerevisiae and W. anomalus CBS5759 yeast (about 290 mg TE/100 mL). Other wines were characterized by lower similar total polyphenol content ranging from 228 up to 269 mg TE/100 mL. The results of the amount of selected polyphenolic compounds detected using HPLC were presented in Table 3. Wines obtained after fermentation by a pure culture of S. cerevisiae (unpasteurized as well as pasteurized musts) were characterized by the highest level of these compounds. It was connected with relatively high concentration of catechin, procyanidins, and phloridzin. Samples produced from pasteurized musts contained almost two times more epicatechin and procyanidins compared to ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

P. Satora et al.

(0.81) (0.49) (0.44) (0.36) (0.39) (0.53) (0.43) (0.68) (0.47)

2.20e 1.00d 0.96d 0.45bc 0.28ab 0.54c 0.27ab 0.81d 0.24a ***

(0.30) (0.14) (0.00) (0.03) (0.07) (0.00) (0.03) (0.03) (0.00)

those from unpasteurized musts. Inoculation of unpasteurized must with W. anomalus yeasts caused an increase in polyphenolic acid concentrations, mainly chlorogenic (16.5 mg L 1) and p-coumaric (1.1 mg L 1). Several fold higher amounts of quercetin galactoside was also found in these samples.

6.74cd 5.78c 6.09bcd 6.68cd 5.20b 6.29d 4.27a 5.23b 4.17a ***

Volatile composition of apple wines

+

+

+

Spontaneous Spontaneous+K4 Spontaneous+K8 JR JR JR+K4 JR+K4 JR+K8 JR+K8 Sig.

ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

Values with different superscript roman letters (a–d) in the same column are significantly different according to the Duncan’s test (P < 0.05). Sig., significance; *, **, *** display the significance at 5%, 1%, and 0.5% by least significant difference; ns, not significant.

(0.10) (0.08) (0.00) (0.00) (0.05) (0.00) (0.02) (0.13) (0.02) 0.29 0.27 0.30 0.32 0.22 0.16 0.23 0.23 0.24 ns (0.54) (0.27) (0.23) (0.39) (0.26) (0.23) (0.17) (0.37) (0.16) 2.37b 1.29a 1.06a 1.54a 1.29a 1.14a 1.12a 0.98a 1.03a *** (0.07) (0.07) (0.08) (0.13) (0.07) (0.08) (0.16) (0.06) (0.05) 0.54 0.36 0.50 0.54 0.55 0.61 0.60 0.42 0.54 ns (0.37) (0.13) (0.10) (0.05) (0.06) (0.25) (0.15) (0.05) (0.15) 1.44 0.97 1.21 1.23 1.02 1.21 1.08 1.04 1.08 ns 1.69b 2.66d 2.75d 2.72d 1.82b 2.76d 1.04a 2.33c 1.07a *** (0.04) (0.02) (0.02) (0.15) (0.04) (0.02) (0.06) (0.03) (0.10) 0,41 0.22 0.26 0.33 0.31 0.42 0.20 0.23 0.20 ns

(0.20) (0.13) (0.01) (0.14) (0.02) (0.04) (0.19) (0.04) (0.11)

Volatile acidity (g L 1) Total organic acids (g L 1) Succinic acid (g L 1) Citric acid (g L 1) Lactic acid (g L 1) Acetic acid (g L 1) Malic acid (g L 1) Tartaric acid (g L 1) Pasteurization Apple wines

Table 2. The organic acids composition of apple wines obtained using spontaneous fermentation as well as Saccharomyces cerevisiae cv. Johannisberg-Riesling (JR) and/or Wickerhamomyces anomalus CBS 1982 (K8) and CBS 5759 (K4) yeast strains

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As with above-mentioned compounds, the volatile composition of obtained apple wines also showed statistically significant variation (Table 4). Most of the volatile components detected, such as acetaldehyde, ethyl acetate, ethyl capronate, the sum of volatile esters as well as propanol, were present at higher concentrations in spontaneously fermented wines. Addition to these musts of W. anomalus strains significantly influenced the volatile profile of apple wines. A reduction in the amount of acetaldehyde, total volatile esters, and propanol was observed; however, the level of other higher alcohols such as isobutanol, amyl alcohols, and 2-phenylethanol increased. The volatile composition of apple wines was strongly changed by S. cerevisiae yeasts. These microorganisms reduced the concentration of acetaldehyde almost 10-fold and of acetone twofold and also reduced ethyl acetate and propanol concentration, but formed higher levels of higher alcohols. Pasteurization of musts also affected the volatile profile of apple wines. It was affirmed that this technological process raised the amount of isoamyl acetate, isobutanol, amyl alcohols, and 2-phenylethanol as well as the sum of volatile esters measured by titration. At the same time, the level of methyl alcohol was decreased. The application of mixed cultures of S. cerevisiae and W. anomalus influenced the volatile composition of apple wines (Table 4), but the differences between samples obtained in the presence of W. anomalus CBS1982 and CBS5759 strains were relatively low. Compared to wines obtained using pure cultures of S. cerevisiae, those produced by mixed cultures contained more acetaldehyde, acetone, and methanol. The level of other analyzed compounds was similar. Sensory evaluation of apple wines

The results of the sensory evaluations of the apple wines are given in Table 5. Sensory evaluation of samples was performed by applying the method of positive rating according to Buxbaum’s model (Tesevic et al., 2005). Statistically significant differences (P = 0.05) were found in the sensory characteristics of examined wines, which was connected with their diverse chemical composition. FEMS Yeast Res 14 (2014) 729–740

Apple wines

+

+

+

Total antioxidant activity (mg TE / 100mL)

67.1a (7.9) 68.1a (11.0) 68.2a (2.6) 62.2a (4.5) 88.7b (2.1) 65.1a (4.8) 82.4b (4.3) 63.7a (4.5) 66.9a (5.0) ***

Total phenol index (mg GAE per L)

323.9c (20.9) 247.0ab (50.8) 256.8ab (11.6) 228.0a (14.8) 268.6ab (5.1) 251.9ab (26.0) 259.0ab (15.3) 291.0bc (25.3) 288.5bc (16.2) ***

Chlorogenic acid (mg L−1) ***

16.5b (0.5)

13.4a (0.5) 13.7a (0.4)

12.7a (0.8)

Caffeic acid glucoside (mg L−1) ***

0.3b (0.1)

0.6c (0.1) 0.2a (0.0)

0.3ab (0.0)

p–Coumaric acid (mg L−1) ***

1.1c (0.1)

0.1a (0.0) 0.4b (0.1)

0.2a (0.0)

p–Coumarylquinic acid (mg L−1) ns

0.3 (0.0)

0.3 (0.1) 0.3 (0.0)

0.3 (0.1)

( + ) catechin (mg L−1) ***

0.3a (0.0)

1.9c (0.1) 2.6d (0.1)

0.9b (0.1)

(–) epicatechin (mg L−1) ***

3.8a (0.4)

6.6b (0.3) 11.4c (0.4)

6.8b (0.2)

Procyanidin B1 (mg L−1) ***

0.2a (0.0)

5.1c (0.2) 9.0d (0.1)

0.9b (0.0)

Procyanidin B2 (mg L−1) ***

2.2a (0.2)

5.1b (0.3) 9.0c (0.1)

2.3a (0.1)

***

0.6a (0.1)

1.6b (0.2) 2.9d (0.2)

2.5c (0.1)

Procyanidin C1 (mg L−1)

Values with different superscript roman letters (a–c) in the same column are significantly different according to the Duncan’s test (P < 0.05). Sig., significance; *, **, *** display the significance at 5%, 1%, and 0.5% by least significant difference; ns, not significant.

Sig.1

JR+K8

JR+K8

JR+K4

JR+K4

JR

Spontaneous +K4 Spontaneous +K8 JR

Pasteurization

FEMS Yeast Res 14 (2014) 729–740

Spontaneous

Phloridzin (mg L−1) ***

0.2a (0.0)

2.3bc (0.3) 2.5c (0.1)

2.1b (0.0)

Phloretin xyloglucoside (mg L−1) ***

0.3a (0.0)

1.0bc (0.1) 1.1c (0.1)

0.9b (0.1)

Quercetin galactoside (mg L−1) ***

2.3b (0.2)

0.5a (0.1) 0.4a (0.0)

0.6a (0.0)

Quercetin glucoside (mg L−1) –

0.0

0.0

0.0

0.1 (0.0)

Quercetin arabinoside (mg L−1) ***

0.6c (0.0)

0.5b (0.1) 0.0a

0.6c (0.0)

Quercetin xyloside (mg L−1) –

0.1 (0.0)

0.2 (0.0) 0.2 (0.0)

0.3 (0.0)

Quercetin rhamnoside (mg L−1) ***

1.9b (0.2)

1.7b (0.1) 0.6a (0.1)

1.8b (0.0)

***

30.7a (1.8)

40.7c (1.3) 54.2d (1.2)

33.3b (1.1)

Total polyphenols (mg L−1)

Table 3. The antioxidant activity and polyphenols profile of apple wines obtained using spontaneous fermentation as well as Saccharomyces cerevisiae cv. Johannisberg-Riesling (JR) and/or Wickerhamomyces anomalus CBS 1982 (K8) and CBS 5759 (K4) yeast strains

Influence of W. anomalus yeast on apple wines composition

735

ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

Apple wines

Pasteurization

+

+

+

Acetaldehyde (mg L−1)

19.3d (2.3) 17.5cd (2.7) 13.2b (1.2) 2.2a (0.2) 2.1a (0.2) 3.0a (0.9) 11.4b (3.3) 10.4b (2.2) 14.4bc (3.8) ***

Acetone (mg L−1) 5.1d (0.6) 4.9d (0.8) 5.2d (0.8) 2.6b (0.7) 1.3a (0.3) 3.0bc (0.8) 4.2cd (0.9) 5.2d (0.3) 4.3d (0.5) ***

Ethyl acetate (mg L−1) 38.5c (1.6) 42.2d (3.6) 37.8c (1.9) 8.2a (2.0) 5.8a (0.8) 8.8a (2.9) 8.0a (0.6) 12.6b (0.5) 7.9a (1.1) ***

Isoamyl acetate (mg L−1) 0.3c (0.1) 0.3c (0.1) 0.2bc (0.0) 0.1ab (0.0) 0.7d (0.1) 0.1ab (0.0) 1.4e (0.1) 0.0a (0.0) 0.0a (0.0) ***

Ethyl capronate (mg L−1) 2.6bc (0.8) 2.8c (0.6) 2.1abc (0.7) 1.6a (0.6) 1.3a (0.2) 1.8ab (0.5) 1.8ab (0.2) 1.4a (0.2) 1.8ab (0.3) **

Sum of volatile esters (mg L−1) 52.4c (4.3) 47.4c (3.0) 40.3b (2.1) 20.1a (4.1) 47.4c (1.0) 15.4a (0.1) 36.6b (2.0) 17.8a (1.9) 36.4b (7.2) ***

32.2bcd (3.8) 36.7cde (2.9) 25.3ab (4.9) 41.8ef (2.2) 18.8a (2.4) 93.3g (9.2) 39.5de (4.4) 49.0f (2.7) 29.6bc (5.3) ***

Methanol (mg L−1)

Values with different superscript roman letters (a–c) in the same column are significantly different according to the Duncan’s test (P < 0.05). Sig., significance; *, **, *** display the significance at 5%, 1%, and 0.5% by least significant difference; ns, not significant.

Sig.

JR+K8

JR+K8

JR+K4

JR+K4

JR

JR

Spontaneous+K8

Spontaneous+K4

Spontaneous

Propanol (mg L−1) 148.4d (11.0) 146.0d (22.9) 130.2bcd (31.2) 133.8cd (26.2) 94.8ab (7.7) 103.3abc (14.9) 98.3abc (24.4) 114.9abcd (15.5) 88.3a (3.4) **

Isobutanol (mg L−1) 151.4a (22.0) 254.7cd (37.4) 240.0c (38.7) 209.4bc (40.1) 237.8c (13.1) 257.6cd (15.5) 265.7cd (36.0) 162.4ab (22.3) 313.2d (44.1) ***

Amyl alcohols (mg L−1) 216.4a (33.7) 507.0f (98.0) 443.2ef (10.9) 260.5ab (30.8) 347.0cd (20.1) 373.8cde (12.2) 372.4cde (11.1) 294.7bc (40.4) 401.3de (53.3) ***

2-Phenylethanol (mg L−1) 32.3a (2.2) 40.2c (1.5) 38.1bc (1.7) 33.9a (1.6) 56.0e (3.4) 36.2ab (1.8) 51.1d (1.2) 34.1a (3.1) 50.7d (2.0) ***

549a (56) 948d (139) 851cd (65) 638ab (69) 736bc (18) 771c (10) 788c (44) 606a (49) 853cd (92) ***

Total fusel alcohols (mg L−1)

Table 4. The volatile composition of apple wines obtained using spontaneous fermentation as well as Saccharomyces cerevisiae cv. Johannisberg-Riesling (JR) and/or Wickerhamomyces anomalus CBS 1982 (K8) and CBS 5759 (K4) yeast strains

736 P. Satora et al.

FEMS Yeast Res 14 (2014) 729–740

Influence of W. anomalus yeast on apple wines composition

737

Table 5. Sensory analysis of apple wines obtained using spontaneous fermentation as well as Saccharomyces cerevisiae cv. Johannisberg-Riesling (JR) and/or Wickerhamomyces anomalus CBS 1982 (K8) and CBS 5759 (K4) yeast strains – Buxbaum model of positive ranking Assessment characteristics Apple wines Spontaneous Spontaneous+K4 Spontaneous+K8 JR JR JR+K4 JR+K4 JR+K8 JR+K8 Sig.1

Pasteurization

+ + +

Color (max 2 pts)

Clearness (max 2 pts)

Odor (max 4 pts)

Taste (max 12 pts)

Total (max 20 pts)

1.5a (0.1) 1.6ab (0.1) 1.7bc (0.1) 1.7cd (0.0) 1.8e (0.1) 1.7cd (0.0) 1.8de (0.0) 1.7cde (0.1) 1.8cde (0.1) ***

1.3a (0.2) 1.3a (0.1) 1.5b (0.1) 1.5bc (0.1) 1.9d (0.1) 1.6c (0.0) 1.8d (0.1) 1.6bc (0.1) 1.8d (0.0) ***

2.2a (0.2) 2.4b (0.1) 2.5bc (0.0) 2.6cd (0.1) 2.8d (0.1) 2.5bc (0.1) 2.7d (0.1) 2.7d (0.1) 2.7d (0.1) ***

8.6a (0.4) 9.1b (0.3) 9.2b (0.2) 10.2d (0.2) 11.8e (0.1) 9.8c (0.1) 11.6e (0.1) 10.3d (0.1) 11.5e (0.1) ***

13.5a (0.5) 14.4b (0.6) 14.8b (0.3) 16.1cd (0.2) 18.3f (0.1) 15.6c (0.2) 18.0ef (0.1) 16.2d (0.1) 17.7e (0.1) ***

Values with different superscript roman letters (a–c) in the same column are significantly different according to the Duncan’s test (P < 0.05). Sig., significance; *, **, *** display the significance at 5%, 1%, and 0.5% by least significant difference; ns, not significant.

Among those studied, the lowest scores were gained by the samples produced by spontaneous fermentation: 13.5 points. Inoculation of spontaneously fermenting musts with killer strains of W. anomalus improved all the analyzed sensory features. According to testers, these wines were characterized by better color and significantly more pleasant and milder taste and aroma. Wines obtained after fermentation with S. cerevisiae yeasts gained very high scores. The samples were distinguished by high clarity and intense apple aroma. Pasteurization of apple musts favorably influenced the sensory features of apple wines. The samples prepared in this way received a few points more compared to those of unpasteurized must. Thermal treatment of must caused deepening of wine color as well as harmonizing of taste that was without any bitterness. Samples after fermentation with mixed cultures of S. cerevisiae and W. anomalus obtained similar scores to those produced by a pure culture of S. cerevisiae. However, the CBS1982 strain more positively modified the sensory profile of apple wines in comparison with CBS5759 yeasts.

Discussion Spontaneous fermentation with microbiota that are present on the fruit surface is used in conventional wine making in different regions of the world (Erten et al., 2006). In the case of apple wines, most often for fermentation specially selected strains of the Saccharomyces genus are applied that during the process produce different compounds influencing the sensory profile of the beverage (Satora et al., 2009). Removal of indigenous microbiota occurred by pasteurization or sulfurization (Downing, 1989) of apple must. Sulfur dioxide used in the second method is, however, unfavorable for the consumer FEMS Yeast Res 14 (2014) 729–740

because it changes the taste and aroma of produced wine, and in higher amounts, it is toxic (Til et al., 1972). An alternative for both methods mentioned above is inactivation of wild yeasts by killer strains that are introduced into the fermentation medium together with Saccharomyces yeasts. In our studies, we applied two strains of W. anomalus yeast producers of K4 (CBS 5759) and K8 (CBS 1982) toxins as well as S. cerevisiae cv. Johannisberg-Riesling strain that was resistant for both killer toxins, generally used for fermentation of white and fruit wines with relatively low content of tannins. Apple wines obtained after spontaneous fermentation were characterized by the highest level of total extract (Table 1), high volatile acidity (Table 2), increased concentration of acetaldehyde, volatile esters (Table 4) including ethyl acetate as well as the lowest amount of ethyl alcohol (Table 1) and higher alcohols (Table 4). They also gained the lowest points during sensory evaluation (Table 5). It is well known that the chemical composition of spontaneously fermented wines is connected with the presence of different species of microorganisms throughout fermentation, mainly non-Saccharomyces yeasts. During the first few days, representatives of Kloeckera/Hanseniaspora, Candida, and other genera are grown (Erten et al., 2006), which biosynthesized mainly esters and acetic acid (Jolly et al., 2006). If their level is excessive, for example, because of usage for fermentation of injured fruits, they can cause different wine faults (Jolly et al., 2006). With an increase in ethanol concentration, the amount of living cells of non-Saccharomyces decreases, and Saccharomyces strains start to dominate. Indigenous S. cerevisiae and/or S. bayanus strains are not such good fermenters as commercial cultures, which could lead to incomplete fermentation of sugars, and lower levels of ethyl alcohol and other components (Satora & Tuszy nski, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

738

2010). In spontaneously fermented samples, because of a higher cell mortality rate, more acetaldehyde can be formed compared with inoculated fermentation (Arellano et al., 2012). Apple wines obtained during our experiments after spontaneous fermentation also contained less malic acid compared with other samples (Table 2). This phenomenon was not connected with malolactic fermentation, because the level of lactic acid in all samples was almost the same; thus, it should be assumed that for such degradation, non-Saccharomyces yeast strains were responsible. These microorganisms more efficiently utilize Lmalic acid than S. cerevisiae; most of them can also assimilate this compound as a sole source of carbon (Saayman & Viljoen-Bloom, 2006). Strains of W. anomalus are considered to be rather weak fermenters, produce up to 35 g L 1 of ethanol (Petrovska et al., 1999), and they are present only at the beginning of fermentation, and as the fermentation progress, they successively die off. Addition of 0.05 g L 1 W. anomalus killer strains to the unpasteurized must significantly changed the fermentation kinetics and chemical composition of apple wines. It happened by modification of the quantitative and qualitative profile of fungal microbiota during first days of fermentation. In these conditions, only killer-resistant strains can grow (Maturano et al., 2012). The strain of W. anomalus CBS 5759 affected stronger indigenous microbiota of apple must compared with CBS 1982 strain. Weakening of the fermentation rate after the 9th day (Fig. 1) and reduced ethanol biosynthesis (Table 1) were found in fermenting must inoculated only with CBS 5759 strain. In these samples, the value of volatile acidity was highly decreased (Table 2), while the amount of higher alcohols (Table 4) and titratable acidity (Table 1) increased; it shows that secreted killer toxins reduce the growth of non-Saccharomyces yeasts. According to Corte-Real & Leao (1990), Wickerhamomyces anomalus yeast can utilize malic acid as the sole carbon source, but this ability is repressed in the presence of sugar. That was the reason that in wines produced with W. anomalus yeasts a higher concentration of L-malic acid than in spontaneously fermented samples was found (Table 2). The opposite tendency was observed in the case of citric acid, because this component, such as maleic acid, malonic acid, oxalic acid, and other Krebs cycle acids, is transported by an accumulative dicarboxylate proton symporter that is not repressed by glucose (Corte-Real & Leao, 1990). Inoculation of must with wine strains of S. cerevisiae which are strong fermenters fundamentally changes the microbiota composition in the must as well as fermentation parameters. It is generally accepted that addition to a must of 106 cells mL 1 of Saccharomyces induces wine fermentation and reduces the possibility of spoilage ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

P. Satora et al.

(Erten et al., 2006). Wines after fermentation with S. cerevisiae strain significantly differed regarding chemical composition compared with those obtained by spontaneous fermentation (Tables 1–4). These samples contained less sugar-free extract, carbonyl compounds, and esters, showed lower titratable and volatile acidity, and were characterized by higher concentration of ethanol and higher alcohols. At the same time, apple wines produced from unpasteurized must differed from those of pasteurized must. Above all, pasteurization increased the fermentation efficiency (Table 1). It could be connected with higher extraction from apple must particles of soluble constituents such as single sugars (Rivas et al., 2005). In wines, after must pasteurization, higher amounts of polyphenol compounds were also detected (Table 2), which also could be associated with higher release of those components to wine as well as with deactivation of apple native enzymes such as polyphenol oxidase (PPO; Chen et al., 2004). Our studies showed a similar tendency to that detected by Aguilar-Rosas et al. (2007) that thermal pasteurization causes a decrease in malic acid concentration in apple musts. However, a decrease was not observed in the case of other analyzed organic acids. Application of W. anomalus strains together with S. cerevisiae yeast as a mixed culture positively influenced the chemical composition and sensory features of produced apple wines. The obtained beverages were characterized by the highest amounts of ethyl alcohol and generally more residual sugars left after fermentation (Table 1). In the earlier studies, it was found that yeast in mixed cultures produces more ethanol than as a pure culture as a result of synergy between strains. More polysaccharides were also detected (Domizio et al., 2011; Maturano et al., 2012). It could be connected with enzymes produced by non-Saccharomyces yeasts and secrete to the medium (for example saccharases etc.), and other components release during lysis of died cells. Wickerhamomyces anomalus is alive during fermentation only when a concentration of ethanol is < 3–4% (Rojas et al., 2003); after death, their cells could be used by S. cerevisiae as a source of carbon, nitrogen compounds, vitamins, sterols, unsaturated fatty acids, and other components. Apple wines obtained with mixed cultures contained more carbonyl compounds, ethyl acetate, and amyl alcohols (Table 4) compared with those after fermentation with monoculture. According to Kurita (2008), the presence of S. cerevisiae yeast in fermenting medium weakened the capacity of W. anomalus for the production of ethyl acetate, at the same time stimulating formation of acetic acid, isoamyl acetate, and isoamyl alcohol. An unfavorable feature of wines obtained using W. anomalus/S. cerevisiae mixed cultures was an increased level of methanol (Table 4). Non-Saccharomyces FEMS Yeast Res 14 (2014) 729–740

Influence of W. anomalus yeast on apple wines composition

yeast may be characterized by higher activity of pectinesterase than S. cerevisiae strains (Blanco et al., 1999); therefore, they can increase the concentration of this compound, especially when for the fermentation, raw material that contains pectins with a higher level of methylation (such as apples) was used (Satora et al., 2009). Apple wines obtained using W. anomalus gained high scores during sensory evaluation; they were equal in quality to samples produced by a pure culture of S. cerevisiae, which together with their antifungal properties confirms the technological usefulness of these yeasts in winemaking. Cultures of W. anomalus can differ one from another by both genetic and metabolic profile as well as by secreted killer toxins (Passoth et al., 2006). Our analysis showed differences between W. anomalus strains used for the fermentation. Each of them in a different way influenced the process of spontaneous fermentation and chemical characteristics of produced apple wines. CBS1982 strain strongly weakened the spontaneous fermentation kinetics (Fig. 1), and apple wines produced after this contained higher amounts of volatile compounds (Table 4). All wines obtained using CBS5759 strain were characterized by higher levels of residual sugars, ethanol, and total polyphenols as well as lower sugarfree extract (Table 1 and 3). It could be connected with secretion of exocellular enzymes such as invertase (Rodrıguez et al., 1995) or b-glucosidase (Restuccia et al., 2011). In the samples, after fermentation with presence of W. anomalus CBS5759, less methanol occurred (Table 4). However, all these differences did not significantly influence the sensory characteristics, and apple wines produced using W. anomalus CBS1982 and CBS5759 were similarly graded (Table 5). In conclusion, application of W. anomalus/S. cerevisiae mixed cultures significantly influenced the fermentation kinetics and chemical composition of apple wines. Inoculation of unpasteurized must only with W. anomalus strain caused correction of sensory features of apple wines, which was probably associated with increasing of volatile acids, acetaldehyde, and ethyl acetate concentration. The apple wines of best quality were obtained after fermentation of pasteurized musts inoculated with S. cerevisiae and W. anomalus CBS5759. The use of W. anomalus enabled increasing degree of attenuation, ethanol concentration, and microbial stability of apple wines without worsening their quality.

Acknowledgements The research was financed in part by Polish Committee for Research in the years 2009–2011 as scientific project No N N312 211336. FEMS Yeast Res 14 (2014) 729–740

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