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Food Microbiology 42 (2014) 218e224

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Food Microbiology

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Selection of appropriate Schizosaccharomyces strains for winemaking

S. Benito*, F. Palomero, F. Calderón, D. Palmero, J.A. Suárez-LepeDept. Tecnología de Alimentos, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Ciudad Universitaria S/N, 28040Madrid, Spain

a r t i c l e i n f o

Article history:Received 10 October 2013Received in revised form15 February 2014Accepted 18 March 2014Available online 4 April 2014

Keywords:SchizosaccharomycesSelective processesUreaPyruvic acidMalic acidWinemaking

* Corresponding author. Tel.: þ34 913363710.E-mail address: santiago.benito@upm.es (S. Benito

http://dx.doi.org/10.1016/j.fm.2014.03.0140740-0020/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

This paper describes the selection of Schizosaccharomyces yeasts with adequate oenological suitabilityand high capacity for the degradation of malic acid. Despite the almost non-existent number of com-mercial strains, the use of this yeast genus has recently been recommended by the International Orga-nisation of Vine and Wine (OIV, in French). Thus, in the present study, a large number ofSchizosaccharomyces strains were isolated using a selective differential medium. Initially, classic pa-rameters of oenological interest for the use of fermentative strains of Saccharomyces cerevisiae (the mostfrequently used type of yeast) were assessed. Only five strains of moderate acetic acid production lowerthan 0.4 g/L were obtained at the end of fermentation. Other, more specific features of this yeast genus’physiology were also studied, including urease activity and the production of pyruvic acid and glycerol.Finally, oenological suitability was determined by comparing selected strains with other Schizosacchar-omyces reference and S cerevisiae control strains. Schizosaccharomyces strains produced 80% less ureacontent, four times higher pyruvic acid levels and 1 g higher glycerol contents than the Saccharomycesreference strains. The results confirmed that it is possible to perform selective processes on microor-ganisms from the genus Schizosaccharomyces using methodology developed in this work to obtain strainsof industrial interest.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Many research groups are currently studying non-Saccharo-myces yeasts (Contreras et al., 2013) due to their unique physio-logical and metabolic properties, which may be advantageous inwinemaking. Yeasts from the genus Schizosaccharomyces showrapid malic deacidification, converting malic acid to ethanol andCO2 (Fleet, 1993, 2000; Gao and Fleet, 1995; Thornton andRodríguez, 1996; Dharmadhikari and Wilker, 1998) that may beuseful in quality winemaking. However, Schizosaccharomyces is notcurrently used because of specific off-flavours commonly associ-ated with their metabolism. Schizosaccharomyces is commonlyisolated fromwines suffering from organoleptic and chemical faultsthrough the appearance of acetic acid, acetaldehyde, acetoin andethyl acetate (Gallander, 1977; Snow and Gallander, 1979;Unterholzner et al., 1988; Yokotsuka et al., 1993; Pitt and Hocking,1999). These undesirable properties may not be found in allstrains. However to date difficulties associated with the isolation of

).

Schizosaccharomyces have prevented the development of a selectiveprocess for a representative population (Benito et al., 2013b).

The use of the genus Schizosaccharomyces in winemaking hasrecently been recommended by the International Code of Oeno-logical Practices (http://www.oiv.int/oiv/info/enplubicationoiv#code/CODE_2013_EN_revised.pdf). However, despite offering theclassic advantages of malic dehydrogenase activity, only one com-mercial strain of Schizosaccharomyces pombe is currently available(http://www.proenol.pt/files/products/ProMalic_09_2008.pdf). Inrecent years, interest in novel, specific uses of the Schizosacchar-omyces genus in modern oenology has increased. One importantfeature of Schizosaccharomyces is its suitability for lees ageingwhich can be attributed to high autolytic release of cell wall poly-saccharides (Palomero et al., 2009). Furthermore, certain Schizo-saccharomyces mutants may be able to reduce the gluconic acidcontent of spoilt musts (Peinado et al., 2007, 2009). The ureaseactivity of Schizosaccharomyces spp. (Deák, 2008; Benito et al.,2013a) is also of interest for food safety. Namely, urease activitymay reduce high ethyl carbamate content in wine by reducing theconcentration of urea (a precursor of ethyl carbamate) (Uthurryet al., 2004). Compared with other species, the elevated produc-tion of pyruvic acid and pyranoanthocyanins (Morata et al., 2012)stabilises the colour of wine.

S. Benito et al. / Food Microbiology 42 (2014) 218e224 219

The present study aimed to develop the first selective processfor using Schizosaccharomyces yeast strains in industrial appli-cations. Classic selection parameters used for traditionalfermentative yeasts of Saccharomyces were applied, along withmore advanced and specific parameters for Schizosaccharomyces.The proposed selective process (Fig. 1) is a viable alternative tothe use of genetically modified Saccharomyces spp. (Pretorius andBauer, 2002; Husnik et al., 2006; Husnik et al., 2007; Liu and Li,2009) and provides similar end results. The use of geneticallymodified (GMO) Saccharomyces spp. is controversial and isrestricted at the industrial level by European legislation (CE No479/2008).

2. Methods and materials

2.1. Isolation of studied strains

In the selection process (Fig. 1), 100 Schizosaccharomyces strainswere isolated using only the selective differential medium forSchizosaccharomyces. As described in the literature (Benito et al.,2013b) the YEPD medium is enriched with actidione (20 mg/L),benzoic acid (350 mg/L) and malic acid (4 g/L). This liquid selectivedifferential medium was used for 14 honeycombs, 21 non-pasteurised organic honeys and 7 concentrated grape musts. Theselected strains were subsequently isolated from selective liquid

Fig. 1. Proposed methodology for the isolation

media where malic acid degradation higher than 50% was detectedby enzymatic analysis after being incubated at 25 �C for 17 days. Thestrains were cultivated on Petri dishes using the same selectivemedia but in solid agar phase according to the methods describedin Fig. 1.

2.2. First phase of Schizosaccharomyces strain selection

Tubes containing 9 mL of sterilised concentrated must (DreamFruits S.A., Quero, Toledo, Spain), which was diluted to 222 g/Lglucose þ fructose (22.7� Brix) and enriched with 4 g/L malic acid(Panreac, Barcelona, Spain) (final pH 3.1), were inoculated with1 mL of YEPD containing 108 CFU/mL determined using a countingchamber (Thomas Scientific, Swedesboro, United States of America)of each of the isolated Schizosaccharomyces strains. To achieve thispopulation, different yeast strains contained in 10 mL glass tubes insolid YEPD medium were transferred to 5 mL volumes of liquidYEPDmedium usingwire loops. These cultures were grown at 25 �Cfor 24 h 100 mL of each yeast suspension was cultivated in 5 mLvolumes of YEPD at 25 �C for 24 h. This procedure was performedsequentially for three times before the final inoculation of 1 mL inthe fermentativemedium. After 17 days of fermentation, enzymaticanalysis was performed for glucoseþ fructose, malic acid and aceticacid as described below. This experiment was performed in tripli-cate for each studied strain out of a total of 100.

and selection of Schizosaccharomyces spp.

S. Benito et al. / Food Microbiology 42 (2014) 218e224220

2.3. Microorganisms used in the second phase ofSchizosaccharomyces strain selection

The yeast strains used in the second selection phase included asreference microorganisms S. pombe 938 and 936 and Saccharo-myces cerevisiae 87 from the collection of the Institute of IndustrialFermentations (IFI, CSIC, Madrid, Spain). The strains S. pombe 15.4,4.5, 31, V1 and V2 were isolated during the first selection phase ofthe study. Along with S. cerevisiae reference strain SC1, these strainsnow belong to the culture collection at the oenology laboratory ofthe Polytechnic University of Madrid.

2.4. Second phase of Schizosaccharomyces strain selection

Microfermentations were performed using 50 mL aliquots ofconcentrated must (Dream Fruits S.A., Quero, Toledo, Spain), whichwere diluted to glucose þ fructose (G þ F) concentrations of 222 g/L, enriched with 3.2 g/L malic acid (Panreac, Barcelona, Spain) (finalpH 3.22) and inoculated with 1 mL of liquid YEPD medium con-taining 108 CFU/mL (determined using a counting chamber) of oneof the above-mentioned yeasts. The initial value of Primary aminonitrogen in themust was 198mg/Lmeasured using a Biosystems kit(http://www.biosystems.es). All fermentations were performed in100-mL flasks, which were sealed with a Muller valve and filledwith 98% H2SO4 (Panreac, Barcelona, Spain) to allow the release ofCO2 and to prevent microbial contamination (Vaughnan-Martiniand Martini, 1999). The temperature was maintained at 25 �C.The fermentations proceeded without aeration, oxygen injection oragitation. All fermentations were performed in triplicate.

The Gþ F, malic acid, acetic acid and pyruvic acid contents of thefermentations were monitored over a period of 21 days. The ureaand glycerol concentrations were determined at the end offermentation as described below.

2.5. Determination of glucose þ fructose, malic acid, acetic acid,pyruvic acid, urea and glycerol

All analyses were conducted using a Y15 Autoanalyser (Bio-systems, Barcelona, Spain). Enzymatic analyses for Gþ F, malic acid,glycerol, acetic acid and urea were performed using kits obtainedfrom Biosystems (http://www.biosystems.es). Pyruvic acid wasdetermined using an appropriate kit (Megazyme, Bry, Ireland).

2.6. Identification by PCR using the 26S rDNA gene

DNA was extracted according to the method of Querol et al.(1992). The D1/D2 domains of the 26S rDNA gene were amplifiedusing the primer pairs NL1/NL4, and conditions described else-where were employed (Kurtzman and Robnett, 1997). In all cases,PCR reactions were performed in volumes of 25 mL, which con-tained 2 mL (10e100 ng) of template DNA, 1 mL of each primer(20 mM), 2.5 mL of 10� PCR buffer, 1 mL of MgCl2 (50 mM), 0.5 mL ofdNTPs (40 mM) and 0.15 mL of Taq DNA polymerase (5 U/mL). PCRproducts were detected in 1% agarose ethidium bromide gels in TAE1� buffer (40 mM tris-acetate and 1.0 mM EDTA). PCR productswere purified using an UltraClean PCR Clean-up kit (Mo Bio,Carlsbad, California, United States of America) and were sequencedin both directions using the same primers employed in the PCRreactions. An ABI 3730xl apparatus (Applied Biosystems) was usedto perform the sequencing reactions. Nucleotide sequences wereanalysed using DNASTAR 7.1 software. All sequences obtained inthe present study were deposited in GenBank.

2.7. Statistical analysis

All statistical analyses were performed using PC Statgraphics v. 5software (Graphics Software Systems, Rockville, MD, USA). Thesignificance was set to p < 0.05 for the ANOVA matrix F value. Themultiple range test was used to compare the means.

3. Results and discussion

3.1. First phase of Schizosaccharomyces strain selection

After fermenting in test tubes for 17 days, the degradation ofmalic acid, a specific feature of Schizosaccharomyces yeasts, wasassessed for the 100 isolated strains, and two basic parameters(residual glucose þ fructose and acetic acid production) used in theclassic selection of fermentative yeasts of the genus Saccharomyceswere also determined (Loira et al., 2012; Suárez-Lepe and Morata,2012). The final concentrations of glucose þ fructose afterfermentation were close to 0 g/L for the majority of the isolates(Fig. 2a), which is in agreement with data reported by other authorsand is indicative of strong fermentative power (Fleet, 2008; Suárez-Lepe et al., 2012). This property would permit the production of drywines similar to those produced traditionally with classic fermen-tative yeasts selected from the Saccharomyces genus.

The degradation of malic acid, which is the primary industrialuse of Schizosaccharomyces yeasts, was nearly 100% in the vastmajority of strains (Fig. 2b). These data are consistent with thoseprovided by other authors, who reported malic acid degradationrates ranging between 75 and 100% (Silva et al., 2003; Benito et al.,2012, 2013a). However, several strains showed malic acid degra-dation rates between 15 and 60%. Such strains may be of interest ifpartial malic deacidification is intended. Out of the 100 initialSchizosaccharomyces strains, only five (15.4, 4.5, 31, V1 and V2)showed moderate final concentrations of acetic acid (<0.4 g/L)(Fig. 2c).

The data obtained in the present study demonstrate the realpossibility of performing selective processes on Schizosacchar-omyces yeasts that meet the basic requirements of the wine in-dustry for the production of quality wines. The first stage ofselection enabled the rapid discrimination of a large portion ofindividual isolates, especially according to their tendency to pro-duce high concentrations of acetic acid (Benito et al., 2012), prior toassessing more specific factors.

3.2. Comparison between preselected Schizosaccharomyces andculture collection strains

Once five Schizosaccharomyces strains showing moderate aceticacid production were selected, other features were studied infurther detail. Microvinifications were performed to verify thepreviously obtained data and to confirm the proper fermentativeability of the preselected strains. In this portion of the study, thepreselected Schizosaccharomyces strains were compared to S. cer-evisiae reference strains 87 and SC1 and to other non-selectedSchizosaccharomyces strains from type culture collections. For allselected Schizosaccharomyces strains and S. cerevisiae referencestrains the final concentrations of acetic acid were approximately0.4 g/L, while the S. pombe reference strains 936 and 938 providedacetic acid concentrations of up to 1 g/L (Fig. 3a.1, b.1, c.1). Therefore,when used as a single dominant strain during fermentation, thereference strains are not suitable for the production of qualitywines (Benito et al., 2012).

The complete degradation of malic acid was observed for mostof the studied Schizosaccharomyces strains (Fig. 3a.2, b.2, c.2). Thenon-selected strain S. pombe 935 (IFI, CSIC) degraded 62.5% of the

Fig. 2. (a) Glucose þ fructose concentrations (g/L); (b) malic acid degradation (%); (c) acetic acid concentration (g/L). Scatterplots, box-and-whisker plots and histograms for themean values of the final concentrations of glucose þ fructose for the 100 studied Schizosaccharomyces strains after 17 days of fermentation.

S. Benito et al. / Food Microbiology 42 (2014) 218e224 221

Fig. 3. Fermentation kinetics of acetic acid (1), malic acid (2) and glucose þ fructose (3) for (a) selected Schizosaccharomyces strains (V1, V2, 31, 4.5 and 15.4); (b) Schizosacchar-omyces spp. reference strains from the collection (935, 936 and 938); (c) Saccharomyces cerevisiae reference strains (87 and SC1).

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initial malic acid content of the must, which indicated that malicacid degradation was strain-dependent. The selected S. cerevisiaeyeasts only degraded 28% of the initial malic acid content in themust. The degradation of glucose þ fructose was faster in theSaccharomyces strains than in the Schizosaccharomyces strains(Fig. 3a.3, b.3, c.3), as previously observed by other authors (Benitoet al., 2012).

3.3. Other specific criteria for the selection of Schizosaccharomycesyeasts

3.3.1. UreaThe urea content of the finishedwineswas less than 0.5mg/L for

Schizosaccharomyces yeasts (Fig. 4). However, the urea content ofwines produced from Saccharomyces strains ranged between 2.2and 2.7 mg/L. The observed differences were attributed to theurease activity of Schizosaccharomyces (Lubbers et al., 1996; Deák,2008; Benito et al., 2013a). A reduction in urea generally leads topotentially lower ethyl carbamate (a toxic compound) contentbecause urea is its main precursor (Uthurry et al., 2006). Therefore,the use of Schizosaccharomyces strains may improve wine safetyfrom a toxicological point of view. Other authors (Medina et al.,2013) reported that different yeast strains affect differentlybiogenic amines levels produced during malolactic fermentation.The Schizosaccharomyces strains used in this study could alsoreduce the possibility of lactic acid bacteria growth by removing all

malic acid (another nutrient source), which decreases the risk ofbiogenic amine formation by these microorganisms (Lonvaud-Funel, 2001; De Fátima et al., 2007; Alcaide-Hidalgo et al., 2007).

3.3.2. Pyruvic acidThe Schizosaccharomyces strains produced more pyruvic acid

than the studied Saccharomyces yeasts (Fig. 5). Strains V2, 31 and938 yielded amaximum pyruvic acid concentration of 0.42 g/L after48 h of fermentation, which is similar to the values obtained inprevious studies (Morata et al., 2012; Benito et al., 2012). Incontrast, the other Schizosaccharomyces strains provided amaximum pyruvic acid content of 0.2 g/L. Pyruvic acid-based se-lection studies on S. cerevisiae showed maximum values rangingbetween 0.06 and 0.13 g/L after 4 days of fermentation (Morata,2004). These values are four times less than those of the presentSchizosaccharomyces strains. The same author reported a strongcorrelation between the amount of pyruvic acid released into themedium and the formation of vitisin A (a pyranoanthocyanin, anatural polyphenol found in grapes) (Morata et al., 2003). Thus,selected strains with high pyruvate production rates such as V2 and31 may be of interest in terms of colour stability.

3.3.3. GlycerolThe glycerol content (Fig. 6) of wines fermented with Schizo-

saccharomyces strains (maximum concentration of 10 g/L) washigher than that of wines produced from S. cerevisiae strains

Fig. 4. Urea concentrations (means � standard deviation) of the final wines for selected Schizosaccharomyces strains (V1, V2, 31, 4.5 and 15.4); Schizosaccharomyces spp. referencestrains from the collection (935, 936 and 938); Saccharomyces cerevisiae reference strains (87 and SC1).

S. Benito et al. / Food Microbiology 42 (2014) 218e224 223

(maximum concentration of 8.8 g/L). The observed differences inglycerol content are statistically significant (p¼ 0.05) and are in linewith the other results, indicating that S. pombe possesses a highlydeveloped glyceropyruvic pathway, which may explain its greaterproduction of pyruvic acid and glycerol compared with other yeasts(Suárez-Lepe et al., 2012).

3.4. Species identification by PCR using the 26S rDNA gene

The sequences of the D1/D2 26S rDNA of the four selectedstrains were 100% identical to 591 bp of the corresponding genesequence of the S. pombe strain NRRL Y-12796 (Accession No.JQ689077). The sequences were deposited in the EMBL Sequence

Fig. 5. Pyruvic acid concentration during the course of alcoholic fermentation for selected Scstrains from the collection (935, 936 and 938); Saccharomyces cerevisiae reference strains (

Database under Accession Nos. HE963292, HE963293, HE963294and HE963295.

4. Conclusion

Based on the promising results of this study, selection processesfor yeasts of the Schizosaccharomyces genus should be carried out.The proposed methodology for the selection of Schizosacchar-omyces strains can be reproduced by yeast-producing companies orresearch centres, previous to final validation phases, where sensoryanalysis and other quality parameters can be further evaluated.

The strains of S. pombe selected here showed some goodwinemaking abilities and displayed specific metabolic features,

hizosaccharomyces strains (V1, V2, 31, 4.5 and 15.4); Schizosaccharomyces spp. reference87 and SC1).

Fig. 6. Glycerol concentrations (means � standard deviation) of the final wines for selected Schizosaccharomyces strains (V1, V2, 31, 4.5 and 15.4); Schizosaccharomyces spp.reference strains from the collection (935, 936 and 938); Saccharomyces cerevisiae reference strains (87 and SC1).

S. Benito et al. / Food Microbiology 42 (2014) 218e224224

such as high glycerol and pyruvic acid production, excellent ureaseactivity and the ability to deacidify musts. These features are allnoteworthy in the production of quality wines.

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