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. 2016 Mar 23;11(3):e0151102. doi: 10.1371/journal.pone.0151102

Selected Schizosaccharomyces pombe Strains Have Characteristics That Are Beneficial for Winemaking

Ángel Benito 1, Daniel Jeffares 2, Felipe Palomero 1, Fernando Calderón 1, Feng-Yan Bai 3, Jürg Bähler 2, Santiago Benito 1,*
Editor: Juan Mata4
PMCID: PMC4805284  PMID: 27007548

Abstract

At present, wine is generally produced using Saccharomyces yeast followed by Oenococus bacteria to complete malolactic fermentation. This method has some unsolved problems, such as the management of highly acidic musts and the production of potentially toxic products including biogenic amines and ethyl carbamate. Here we explore the potential of the fission yeast Schizosaccharomyces pombe to solve these problems. We characterise an extensive worldwide collection of S. pombe strains according to classic biochemical parameters of oenological interest. We identify three genetically different S. pombe strains that appear suitable for winemaking. These strains compare favourably to standard Saccharomyces cerevisiae winemaking strains, in that they perform effective malic acid deacidification and significantly reduce levels of biogenic amines and ethyl carbamate precursors without the need for any secondary bacterial malolactic fermentation. These findings indicate that the use of certain S. pombe strains could be advantageous for winemaking in regions where malic acid is problematic, and these strains also show superior performance with respect to food safety.

Introduction

Several research teams are studying the winemaking potential of non-Saccharomyces yeast strains [1]. For example, fission yeasts from the genus Schizosaccharomyces show more rapid malic acid deacidification, by converting malic acid to ethanol and CO2 [2–4]. Schizosaccharomyces pombe possesses several remarkable metabolic properties that may be useful in modern quality winemaking [2,5–6], including a malic dehydrogenase activity, high autolytic polysaccharides release [7], ability of gluconic acid reduction [8–11], urease activity [12,13], elevated production of pyruvic acid and colour improvement [14,15], as well as low production of biogenic amines and ethyl carbamate [6,16].

However, S. pombe type strains are not currently employed because of specific off-flavours commonly associated with their metabolism. Indeed, Schizosaccharomyces strains are commonly isolated from wines suffering from strong organoleptic and chemical deviations including the appearance of acetic acid, acetaldehyde, acetoin and ethyl acetate [17–21]. These undesirable properties of the commonly-used strains have been assumed to be general properties of the species, and only one commercial strain of S. pombe is currently available on the market [22]. The exploration of the winemaking properties of other S. pombe strains has generally been overlooked. This omission partly reflects the absence of any specific processes to select strains that are appropriate for winemaking [5], and difficulties with isolating S. pombe from environmental samples [23]. Given this situation, it has been difficult to obtain a representative strain collection of this species. However, guided by a recent analysis of genetic and phenotypic diversity of 161 S. pombe strains [24], we can now conduct a more extensive survey of the utility of this species for winemaking.

The present study examines the winemaking potential of 75 S. pombe strains that are available from different microorganism type culture collections, or have been collected by us [5,25]. We apply classic selection parameters used in traditional fermentative Saccharomyces yeasts, along with more advanced and specific parameters for S. pombe. The results indicate that at least three S. pombe strains can potentially provide a viable and attractive alternative to the application of genetically modified Saccharomyces species [26–29] whose use is restricted in food industry by European legislation [30].

Materials and Methods

Microorganisms

The following yeasts were used for the experimental fermentations: 75 S. pombe strains, including the non-clonal 54 strains recently described [24], the three mating types of the standard laboratory strain: Leupold’s 972 (h−), 975 (h+) and 968 (h90) (JB22, JB32 and JB50), strain V1 collected by us [5], and 17 strains collected from traditional Chinese breweries by Feng-Yan Bai (unpublished). The following S. cerevisiae strains were used as controls: 87 and 88 from the Spanish Type Culture collection, which have been used in previous studies. Data for all strains are provided in Table 1.

Table 1. Schizosaccharomyces pombe yeast strains used in the experiments.

Bähler laboratory strain name strain ID(s) location collected substrate date collected reference
JB4 CBS5557;CCY44-6-1;CBS10393;DBVPG6275;JCM8262;NBRC1608;NCYC683 Spain Listan grapes ND NA
JB22 Leupolds 972 (h-);CBS10395;NCYC1430 France rotten grapes 1947 NA
JB32 Leupolds 975 (h+) NA
JB50 Leupolds 968 (h90) France ND ND NA
JB758 NOTT30;NCYC936;CBS10394 Sri Lanka fermenting toddy 1979 PMID:25665008
JB759 NOTT33;Y0036;CBS10498 South Africa beverage ND PMID:25665008
JB760 NOTT50;DBVPG2812 Italy (Sicily) grape must treated with SO2 1966 PMID:25665008
JB762 NOTT75;CBS358;DBVPG6374 ND ND 1987 PMID:25665008
JB837 NOTT1;UWOPS92.229.4 Mexico Tequilla 1992 PMID:25665008
JB838 NOTT2;UWOPS94.422.2 Mexico Tequilla 1994 PMID:25665008
JB840 NOTT4;UFMGR435;CBS10458 Brazil (Aracaju) frozen pulp of Eugenia uniflora ("pitanga", Myrtaceae) 1999 PMID:25665008
JB841 NOTT5;UFMGA1263;CBS10469 Brazil (Vicosa) must of Brazilian cachaça 1996 PMID:25665008
JB842 NOTT6;UFMGA602;CBS10460 Brazil (Belo Horizonte) must of Brazilian cachaça 1996 PMID:25665008
JB845 NOTT9;UFMGR434;CBS10476 Brazil (Aracaju) frozen pulp of Eugenia uniflora ("pitanga", Myrtaceae) 1999 PMID:25665008
JB846 NOTT10;UFMGA826;CBS10465 Brazil (Belo Horizonte) must of Brazilian cachaça 2000 PMID:25665008
JB848 NOTT12;UFMGR428;CBS10475 Brazil (Aracaju) frozen pulp of Eugenia uniflora ("pitanga", Myrtaceae) 1999 PMID:25665008
JB852 NOTT16;UFMGA529;CBS10462 Brazil (Belo Horizonte) must of Brazilian cachaça 1996 PMID:25665008
JB853 NOTT17;UFMGA1000;CBS10465 Brazil (Belo Horizonte) must of Brazilian cachaça 1996 PMID:25665008
JB854 NOTT18;UFMGR427;CBS10474 Brazil (Aracaju) frozen pulp of Eugenia uniflora ("pitanga", Myrtaceae) 1999 PMID:25665008
JB858 NOTT22;UFMGA738;CBS10464 Brazil (Belo Horizonte) must of Brazilian cachaça 1996 PMID:25665008
JB862 NOTT26;NCYC380;CBS10392 ND raw cane sugar 1953 PMID:25665008
JB864 NOTT28*;ATCC24751;CBS10391;NCYC132 Africa African Millet Beer 1921 PMID:25665008
JB870 NOTT34;Y0037;CBS10499 South Africa wine ND PMID:25665008
JB872 NOTT36;CBS2775;IFO0347;NBRC0347;NCYC3418 Japan fermenting molasses 1957 PMID:25665008
JB873 NOTT37;CBS5680;DBVPG6448;NCYC3422 Poland apple 1965 PMID:25665008
JB874 NOTT38;CBS5682;DBVPG6376;NCYC3421 South Africa millet beer 1965 PMID:25665008
JB875 NOTT39;CBS7335 Spain alpechín (water which oozes from a heap of olives) 1988 PMID:25665008
JB878 NOTT42;DBVPG2804 Malta wine 1963 PMID:25665008
JB879 NOTT43;DBVPG2805 Malta wine 1963 PMID:25665008
JB884 NOTT48;DBVPG2810 Malta wine 1963 PMID:25665008
JB899 NOTT63;Y470? ND ND ND PMID:25665008
JB900 NOTT64;Y831 South Africa industrial glucose ND PMID:25665008
JB902 NOTT66;CBS374 Netherlands (Delft) molasses 1928 PMID:25665008
JB910 NOTT74;DBVPG6449;CBS1062;IFO0344;NRRLY-1358 ND cane sugar molasses ND PMID:25665008
JB913 NOTT77;CBS1058 Indonesia molasses 1949 PMID:25665008
JB914 NOTT78;CBS357;DBVPG6280;CLIB834 Jamaica molasses 1912 PMID:25665008
JB916 NOTT80;CBS352;DBVPG6373 Indonesia arrack factory (distilled alcoholic drink made from sap of the coconut flower) 1923 PMID:25665008
JB917 NOTT81;CBS1057;DBVPG6375 Sweden brewer's yeast 1933 PMID:25665008
JB918 NOTT82;CBS1059 Mauritius cane sugar 1949 PMID:25665008
JB929 NOTT93;CBS1063;DBVPG6450;NRRLY-1362 ND cane sugar molasses 1934 PMID:25665008
JB930 NOTT94;FRR2208 Australia (Nundah) glace syrup ND PMID:25665008
JB931 NOTT95;FRR2535 ND raspberry cordial concentrate 1983 PMID:25665008
JB934 CLIB837 France winemaking ND PMID:25665008
JB938 CLIB841 France winemaking ND PMID:25665008
JB939 CLIB842 Spain winemaking ND PMID:25665008
JB942 CLIB845 France winemaking ND PMID:25665008
JB943 CLIB846 France winemaking ND PMID:25665008
JB953 PHAFF65-116 Australia exudate of a Eucalyptus? Tree ND PMID:25665008
JB1110 YHL266;Zimmer1987_0209 ND red current jelly 1977 PMID:25665008
JB1117 YHL281;Zimmer1987_0208 Holland malt 1975 PMID:25665008
JB1154 AWRI1875 Australia (Barossa Valley) ND ND PMID:25665008
JB1171 CECT12622;NOTT123;IFI356 ND grape juice 1982 PMID:25665008
JB1174 CECT12918;NOTT126;IFI2139 ND grape juice 1982 PMID:25665008
JB1180 Kambucha_YFS276;NOTT132 ND ND ND PMID:25665008
JB1197 NBRC0340;NOTT159;AJ14275;IFO0340;JCM1846 ND ND 1983 PMID:25665008
JB1205 NBRC10568;NOTT167 ND ND ND PMID:25665008
JB1206 NBRC10569;NOTT168 ND ND ND PMID:25665008
JB1207 NBRC10570;NOTT169 ND ND ND PMID:25665008
JB1468 GZLJ3.34 Guizhou Laojiao Distillery, Changshun, Guizhou Dec. 2012 NA
JB1508 Santiago Benito V1; HE963293 Spain Organic Honey 2012 PMID:24929740
JB1469 GZLJ3.36 Guizhou Laojiao Distillery, Changshun, Guizhou Dec. 2012 NA
JB1470 GZLJ3.41 Guizhou Laojiao Distillery, Changshun, Guizhou Dec. 2012 NA
JB1471 GZLJ5.6 Guizhou Laojiao Distillery, Changshun, Guizhou Dec. 2012 NA
JB1472 GZLJ5.28 Guizhou Laojiao Distillery, Changshun, Guizhou Dec. 2012 NA
JB1473 QT11.6 Gucheng Laojiao Distillery, Qitai, Xinjiang Sept. 2013 NA
JB1474 XEBLK30.8 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1475 XEBLK1.1 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1476 XEBLK2.1 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1477 XEBLK3.1 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1478 XEBLK4.1 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1479 XEBLK7.1 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1480 XEBLK22.2 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1481 XEBLK27.3 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1482 XEBLK29.10 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1483 XEBLK30.2 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
JB1484 XEBLK31.1 Xiaoerbulake Distillery, Xinyuan, Xinjiang Sept. 2013 NA
- CECT1375;IFI935 Spain 1983
- CECT1376; IFI936 Spain 1983
- CECT12512; IFI87 Spain must 1999
- CECT12513; IFI88 Spain must 1999
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First phase of S. pombe strain selection

Starter cultures were grown from 100 μL of each yeast suspension, cultivated in 5 mL volumes of YEPD at 25°C for 24 h. This procedure was performed in triplicate before the final inoculation of 1 mL in the fermentative medium. 1 mL (108 CFU/mL) of these starters cultures were then inoculated into tubes containing 9 ml of sterilised concentrated must (Dream Fruits S.A., Quero, Toledo, Spain), which was diluted to 212 g/L glucose + fructose and enriched with 4 g/L malic acid (Panreac, Barcelona, Spain) (final pH 3.1) to simulate acidic musts where the use of S. pombe is more recommended in order to increase wine quality [3,5]. After 21 days of fermentation, enzymatic analysis was performed for glucose + fructose, malic acid and acetic acid (S1 Table). This experiment was performed in triplicate for each studied strain.

Yeasts used in the second phase of S. pombe strain selection

The yeast strains used in the second selection phase included S. pombe IFI936/CECT12774 and IFI935/CECT1376 and S. cerevisiae IFI87/CECT12512 and IFI88/CECT12513 from the Spanish Type Culture collection. The S. pombe strains JB899/Y470, JB873/NCYC3422 and JB917/CBS1057 were selected during the first phase of the study. The strain V1 was selected in a previous study [5].

Second phase of S. pombe strain selection

Microfermentations were performed using 200 ml aliquots of concentrated must (Dream Fruits S.A., Quero, Toledo, Spain), which were diluted to glucose + fructose (G + F) concentrations of 211 g/L, enriched to 4.3 g/L malic acid (Panreac, Barcelona, Spain) (final pH 3.08), with 60 g/L of Actimax Natura (Agrovín S.A., Ciudad Real, Spain) added to provide nutrition. Each microfermentator was inoculated with 1 mL of liquid YEPD medium containing 107 CFU/mL (determined using a Thomas chamber) of one of the above-mentioned yeasts. All fermentations were performed in 250-ml flasks, which were sealed with a Muller valve and filled with 98% H2SO4 (Panreac, Barcelona, Spain) to allow the release of CO2 and to prevent microbial contamination [31]. The temperature was maintained at 25°C. The fermentations proceeded without aeration, oxygen injection or agitation. All fermentations were performed in triplicate.

The glucose + fructose, malic acid, acetic acid and pyruvic acid contents of the fermentations were monitored over a period of 14 days. The pH, urea, citric acid, glycerol, alcohol content, volatile compounds, amino acids and biogenic amine concentrations were determined at the end of the fermentation.

Measurements of biochemical compounds and pH

Glucose + fructose, malic acid, L-lactic acid, acetic acid, pyruvic acid, urea, glycerol and pyruvic acid analyses (Table 2) were conducted using a Y15 Autoanalyser (Biosystems, Barcelona, Spain). The kits to perform the analyses were obtained from Biosystems [32]. Alcohol content was determined by using the boiling method GAB Microebu [33]. The pH was measured with a Crison pH Meter Basic 20 (Crison, Barcelona, Spain).

Table 2. Final analysis of fermentations performed by the studied strains.

Selected strains are JB899/Y470, JB917/CBS1057, JB873/NCYC3422 and V1, non-selected S. pombe strains from the Spanish culture collection (IFI936/CECT12774 and IFI935/CECT1376) and selected S. cerevisiae strains (IFI87/CECT12512 and IFI88/CECT12513).

Compounds 899 917 873 V1 935 936 87 88
l-Lactic Acid (g/L) 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a
l-Malic Acid (g/L) 0.02 ± 0.01a 0.01 ± 0.01a 0.02 ± 0.01a 0.01 ± 0.01a 1.02 ± 0.11b 0.01 ± 0.01a 3.16 ± 0.14c 3.72 ± 0.08d
Acetic Acid (g/L) 0.24 ± 0.02a 0.29 ± 0.02b 0.33 ± 0.03b 0.30 ± 0.02b 0.93 ± 0.05c 0.98 ± 0.07c 0.21 ± 0.02a 0.24 ± 0.02a
Residual Sugar (g/L) 1.77 ± 0.52a 1.89 ± 0.64a 2.02 ± 0.58a 2.12 ± 0.36a 2.11 ± 0.43a 1.99 ± 0.35a 1.74 ± 0.26a 2.22 ± 0.31a
Glycerol (g/L) 8.32 ± 0.16a 8.88 ± 0.21c 8.02 ± 0.09a 8.91 ± 0.18c 8.14 ± 0.13a 8.48 ± 0.11b 8.36 ± 0.14ab 8.14 ± 0.08a
pH 3.44 ± 0.02d 3.45 ± 0.02d 3.44 ± 0.02d 3.42 ± 0.02d 3.36 ± 0.03c 3.45 ± 0.03d 3.14 ± 0.02 b 3.11 ± 0.02a
Urea (mg/L) 0.42 ± 0.02a 0.48 ± 0.02b 0.39 ± 0.03a 0.44 ± 0.02ab 0.42 ± 0.03a 0.51 ± 0.03b 2.48 ± 0.03c 2.52 ± 0.04c
Citric Acid (g/L) 0.27 ± 0.01a 0.29 ± 0.02a 0.29 ± 0.03a 0.28 ± 0.02a 0.28 ± 0.03a 0.27 ± 0.02a 0.29 ± 0.02a 0.28 ± 0.01a
Alcohol (% v/v) 12.15 ± 0.02b 12.04 ± 0.02a 12.26 ± 0.03c 12.02 ± 0.02a 12.24 ± 0.02c 12.13 ± 0.03b 12.43 ± 0.02d 12.44 ± 0.03d
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Results are the mean ± SD of three replicates. Means in the same row with the same letter are not significantly different (p < 0.05).

Quantification of volatile compounds

Volatile compounds (Table 3) were quantified by headspace gas chromatography–mass spectrometry (HS-GC-MS). Analyses were carried out using a Perkin-Elmer Clarus 500 gas chromatograph with a flame ionization detector, coupled to a mass spectrometer single quadrupole Clarus 560 S, all coupled to an automatic headspace sampler Turbomatrix 110 Trap (Perkin-Elmer, Massachusetts, USA). The headspace sampler conditions were: temperature of thermostating: 80°C, time of thermostating: 45 min, type of trap: Tenax TA, cycles of purge and trap: 4, temperature of trap capture: 45°C, desorption temperature of the trap: 290°C, time of dry trap purge: 10 min, desorption time of trap: 2 min, trap cleaning time: 5 min, needle temperature: 110°C, temperature of HS-GC transfer line: 150°C, vial pressure: 30 psi and constant pressure column: 28 psi. A FFAP capillary column (60 m × 0.25 mm DI x 0.25 μm film thickness) was used. Helium (Air Liquide, España) was used as carrier gas. Gradient analysis was run using the following temperature program: 40°C (3 min); 40–80°C (2°C/min); 80–180°C (3°C/min); and 210°C (5 min). Identification of individual compounds was based on a comparison of the obtained mass spectra of the individual chromatographic peaks with those valid for the standards and available from the National Institute of Standards and Technology (Gaithersburg, MD) software library. We also compared the retention times valid for individual peaks from the wine samples with those of the known volatile components use as standard patterns. To this effect, we used Gas chromatography quality compounds as the sets of the volatile standards (Fluka, Sigma–Aldrich Corp., Buchs SG, Switzerland).

Table 3. Final analysis of volatile compounds performed by the studied strains during fermentations.

Selected S. pombe strains (JB899/Y470, JB917/CBS1057, JB873/NCYC3422 and V1), non-selected S. pombe strains from the Spanish culture collection (936/CECT12774 and IFI 935/CECT1376) and selected S. cerevisiae strains (IFI 87/CECT12512 and IFI 88/CECT12513) from the Spanish culture collection.

Compounds (mg/L) 899 917 873 V1 935 936 87 88
Acetaldehyde 16.46±1.52a 17.82±2.31a 16.99±1.78a 18.13±2.42a 32.46±3.11b 36.52±2.97b 16.18±1.86a 17.56±2.06a
Ethyl lactate 6.32±0.43a 6.58±0.47a 6.52±0.49a 6.39±0.45a 7.76±0.58b 7.82±0.54b 7.23±0.48ab 7.54±0.38b
Ethyl acetate 16.32±1.87a 18.11±2.16a 18.33±2.28a 18.62±2.39a 82.41±4.54b 89.13±5.26b 17.45±1.54a 19.23±3.06a
Diacetyl 2.24±0.16a 2.67±0.32a 2.48±0.26a 2.32±0.24a 2.91±0.55a 2.84±0.48a 2.29±0.33a 2.36±0.38a
Isoamyl acetate 2.28±0.28a 2.21±0.32a 2.44±0.43a 2.33±0.28a 2.68±0.42a 2.59±0.38a 3.77±0.51b 3.62±0.48b
2- Phenyl ethyl acetate 5.25±0.21a 5.36±0.28a 5.29±0.25a 5.38±0.19a 5.48±0.25a 5.54±0.27a 6.41±0.28b 6.34±0.26b
1-Propanol [mg/L] 11.42±1.52a 10.12±1.36a 13.26±2.08ab 10.73±1.84a 18.99±3.62b 19.76±3.93b 28.45±4.01c 31.33±4.13c
Isobutanol 9.31±1.46a 8.57±1.88a 9.86±1.74a 8.94±2.02a 12.11±2.36a 13.52±2.58ab 17.51±2.24b 18.13±2.48b
1-Butanol 5.13±1.03a 5.45±1.09a 5.36±1.14a 5.28±1.19a 7.23±1.26ab 7.44±1.42ab 7.81±1.12b 7.96±1.23b
3-Methyl-butanol 11.36±1.56a 12.16±1.36a 11.98±1.43a 12.28±1.86a 18.44±1.68b 18.88±1.95b 26.12±2.33c 28.06±2.54c
2-Methyl-butanol 23.34±2.36a 24.48±2.43a 25.68±2.67a 24.19±1.88a 31.08±2.09b 33.16±2.24b 40.44±3.16c 42.83±4.23c
Isobutyl acetate n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Ethyl butyrate n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
2-Phenyl-ethanol 21.76±2.11a 22.28±1.98a 21.44±1.78a 22.35±2.23a 24.17±1.79a 24.66±1.68a 28.97±1.82b 29.12±1.96b
Hexanol 1.31±0.12a 1.39±0.14a 1.36±0.15a 1.34±0.16a 1.42±0.14a 1.46±0.16a 2.45±0.21b 2.48±0.26b
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Results are the mean ± SD of three replicates. Means in the same row with the same letter are not significantly different (p < 0.05), n.d. = not detected.

Quantification of biogenic amines

The studied biogenic amines (Table 4) were analysed using a Jasco (Tokyo, Japan) UHPLC chromatograph series X-LCTM, equipped with a Fluorescence detector 3120-FP. Gradients of solvent A (methanol/acetonitrile, 50:50, v/v) and B (sodium acetate /tetrahydrofuran, 99:1, v/v) were used in a C18 (HALO, USA) column (100 mm × 2.1 mm; particle size 2.7 μm) as follows: 60% B (0.25 ml/min) from 0 to 5 min, 60–50% B linear (0.25 ml/min) from 5 to 8 min, 50% B from 8 to 9 min, 50–20% B linear (0.2 ml/min) from 9 to 12 min, 20% B (0.2 ml/min) from 12 to 13 min, 20–60% B linear (0.2 ml/min) from 13 to 14.5 min, and re-equilibration of the column from 14.5 to 17 min. Detection was performed by scanning in the 340–420 nm range. Quantification was performed by comparison against external standards of the studied amines. The different amines were identified by their retention times.

Table 4. Final analysis of biogenic amines.

Fermentations by selected S. pombe strains (JB899/Y470, JB917/CBS1057, JB873/NCYC3422 and V1), non-selected S. pombe strains from the Spanish culture collection (936/CECT12774 and IFI 935/CECT1376) and selected S. cerevisiae strains (IFI 87/CECT12512 and IFI 88/CECT12513).

Compounds 899 917 873 V1 935 936 87 88
Histamine (mg/L) 0.32 ± 0.03a 0.36 ± 0.04a 0.32 ± 0.04a 0.34 ± 0.03a 0.38 ± 0.04a 0.36 ± 0.02a 0.33 ± 0.02a 0.32 ± 0.03a
Tiramine (mg/L) 0.16 ± 0.01a 0.18 ± 0.04a 0.16 ± 0.02a 0.17 ± 0.06a 0.18 ± 0.04a 0.16 ± 0.02a 0.16 ± 0.02a 0.15 ± 0.02a
Phenylethylamine (g/L) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Putrescine (g/L) 0.43 ± 0.03a 0.46 ± 0.06a 0.42 ± 0.05a 0.44 ± 0.04a 0.43 ± 0.05a 0.45 ± 0.07a 0.42 ± 0.03a 0.41 ± 0.04a
Cadaverine (g/L) 0.56 ± 0.03ab 0.55 ± 0.05ab 0.49 ± 0.05a 0.52 ± 0.04a 0.52± 0.03a 0.53 ± 0.03a 0.63 ± 0.03b 0.62 ± 0.04b
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Results represent the mean ± SD for three replicates. Means in the same row with the same letter are not significantly different (p < 0.05), n.d. = not detected.

Analytical determinations of amino acids

Selected amino acids (Table 5) were analysed using a Jasco (Tokyo, Japan) UHPLC chromatograph series X-LCTM, equipped with a Fluorescence detector 3120-FP. Gradients of solvent A (methanol/acetonitrile, 50:50, v/v) and B (sodium acetate /tetrahydrofuran, 99:1, v/v) were used in a C18 (HALO, USA) column (100 mm × 2.1 mm; particle size 2.7 μm) as follows: 90% B (0.25 mL/min) from 0 to 6 min, 90–78% B linear (0.2 mL/min) from 6 to 7.5 min, 78% B from 7.5 to 8 min, 78–74% B linear (0.2 mL/min) from 8 to 8.5 min, 74% B (0.2 mL/min) from 8.5 to 11 min, 74–50% B linear (0.2 mL/min) from 11 to 15 min, 50% B (0.2 mL/min) from 15 to 17 min, 50–20% B linear (0.2 mL/min) from 17 to 21 min, 20–90% B linear (0.2 mL/min) from 21 to 25 min, and re-equilibration of the column from 25 to 26 min. Detection was performed by scanning in the 340–455 nm range. Quantification was performed by comparison against external standards of the studied amino acids. The different amino acids were identified by their retention times.

Table 5. Final analysis of amino acids.

Fermentations by selected S. pombe strains (JB899/Y470, JB917/CBS1057, JB873/NCYC3422 and V1), non-selected S. pombe strains from the Spanish culture collection (936/CECT12774 and IFI 935/CECT1376) and selected S. cerevisiae strains (IFI 87/CECT12512 and IFI 88/CECT12513) from the Spanish culture collection.

Compounds (mg/L) 899 917 873 V1 935 936 87 88
Aspartic acid 10.82±1.51b 11.96±1.68b 11.42±1.33b 12.16±1.78b 21.52±2.46c 18.83±2.13c 7.36±0.68a 8.12±0.74a
Asparagine 19.68±1.56b 20.25±1.72b 19.93±1.84b 21.37±2.12b 30.18±2.89c 29.06±2.52c 16.34±1.09a 17.45±1.14a
Serine 6.25±0.89b 6.88±1.02b 6.47±0.78b 7.43±0.93b 11.69±1.36c 10.78±1.27c 3.18±0.35a 3.64±0.48a
Histidine 81.32±3.59a 83.29±3.52a 82.94±2.89a 84.32±3.46a 92.35±4.48c 94.88±4.69c 65.47±3.18a 69.34±3.46a
Glycine 28.42±1.33a 27.83±1.16a 28.48±1.26a 29.68±1.44a 39.68±1.77b 41.12±2.24b 27.43±1.11a 28.56±1.23a
Arginine 66.52±4.62b 64.52±3.96b 63.14±3.54b 69.52±5.43b 78.21±6.22c 76.52±5.84c 42.43±3.53a 40.88±3.17a
Threonine 53.17±3.46c 51.19±2.98c 46.23±2.72c 49.94±2.56c 37.46±2.97b 36.82±2.64b 24.72±1.82a 22.58±2.01a
Alanine 27.31±2.07a 28.43±2.16a 32.05±3.08a 29.86±3.17a 42.57±3.21b 43.26±3.45b 31.68±2.49a 29.72±2.28a
Tyrosine 9.82±0.68b 10.13±0.62b 11.28±0.86bc 10.22±0.75b 12.88±0.93c 13.31±1.14c 4.48±0.26a 4.69±0.34a
Valine 4.73±0.31c 5.29±0.44c 4.82±0.35c 5.18±0.42c 2.96±0.27b 3.18±0.31b 1.25±0.19a 1.14±0.16a
Tryptophan 1.41±0.26b 1.46±0.22b 1.59±0.28b 1.54±0.23b 1.76±0.23b 1.81±0.29b 0.52±0.13a 0.46±0.07a
Phenylalanine 5.54±0.42b 5.26±0.38b 5.69±0.51b 5.89±0.54b 7.62±0.86b 7.98±1.02 b 3.65±0.29a 3.78±0.33a
Isoleucine 12.04±0.74c 12.56±0.78c 12.64±0.76c 13.24±0.88c 8.17±0.63b 8.35±0.68b 2.45±0.32a 2.62±0.36a
Leucine 15.18±0.93c 14.69±0.87c 15.92±1.05c 15.86±0.92c 10.22±0.81b 11.46±0.98b 3.87±0.68a 3.98±0.673a
Ornithine 18.36±1.65a 17.87±1.42a 18.45±1.72a 19.88±1.78a 19.93±1.86a 20.98±1.89a 29.48±2.27b 30.74±2.36b
Lysine 12.27±0.96b 12.59±0.88b 12.72±0.91b 14.13±1.02b 23.56±1.94c 24.56±2.23c 6.16±0.85a 6.77±0.94a
Methionine 2.68±0.43b 2.89±0.36b 2.73±0.41b 3.27±0.52b 6.26±0.89c 6.47±0.97c 1.23±0.19a 1.29±0.22a
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Results represent the mean ± SD for three replicates. Means in the same row with the same letter are not significantly different (p < 0.05).

Fermentation in stress conditions

Study of fermentation in stress caused by ethanol, sulphur dioxide and the osmotic stress caused by salts (NaCl and KCl), were performed based on methods described previously for S.cerevisiae [34]. 15 ml sealed test tubes contatining 10 ml of sterilized grape must were inoculated with the studied strains. The grape must final sugar concentration was 206 g/L diluted from a concentrated grape must (Dream Fruits S.A., Quero, Toledo, Spain) that was corrected to the respective stressful conditions before yeast inoculation: Ethanol (6%; 10%; 12% and 14%), KCl (0.75 M), NaCl (1.5 M) (both osmotic stress) and KHSO3 (150 and 300 mg/L) (sulphur dioxide). Ethanol, KCl, NaCl products were from Panreac (Panreac, Barcelona, Spain) and KHSO3 was from Agrovín S.A. (Alcázar de San Juan, Spain). The testing tubes were inoculated to a cellular density of 5x106 cells/mL with the studied strains: S. pombe strains (JB899/Y470, JB917/CBS1057, JB873/NCYC3422, JB4/ NCYC683, JB837/ NOTT1 and V1), non-selected S. pombe strains from the Spanish culture collection (936/CECT12774 and IFI 935/CECT1376) and selected S. cerevisiae strains (IFI 87/CECT12512 and IFI 88/CECT12513) from the Spanish culture collection. Final optical density was determined at 640 nm after 24 h of incubation at 25°C by an Y15 spectrophotometer (Biosystems, Barcelona, Spain). The maximal ethanol production was estimated through the fermenting power method [35,36]. In this case the original concentrated grape must (Dream Fruits S.A., Quero, Toledo, Spain) was diluted to a sugar concentration of 300 g/L, the sealed flasks with Müller trapflasks (Alamo, Madrid, Spain) were incubated at 25°C. All the tests were performed in triplicate.

Sensory analysis

For a sensory analysis, fermentations triplicate with S. pombe strains (JB899/Y470, JB873/NCYC3422, JB917/CBS1057, JB4/NCYC683, JB837/ NOTT1, 936/CECT12774, IFI935/CECT1376 and V1) and S. cerevisiae strains (IFI 87/CECT12512 and IFI88/CECT12513) were performed with microvinifications with similar methodology to previously described [13, 15]. Sealed 1 L flasks with a Müller trapflasks (Alamo, Madrid, Spain) containing 800 ml of sterilised (115°, 15 min) concentrated must (Dream Fruits S.A., Quero, Toledo, Spain), which was diluted to 202 g/L glucose + fructose and enriched with 4 g/L malic acid (Panreac, Barcelona, Spain) (final pH 3.11). The flasks were inoculated with the studied strains to initial population of 106 CFU/mL. The completion of the fermentation process was verified by weight loss and confirmation that final enzymatic analysis of glucose + fructose was below 3 g/L after fermenting at 25°C. After fermentation, wines were racked and stored for 7 days at 4°C in 750 mL wine bottles. The final product was bottled in 350 mL wine bottles, sealed bottles and stored horizontally in a climate chamber at 4°C for two weeks until sensory evaluation.

Final wines were assessed (in a blind test) by a panel of 10 experienced wine tasters, all members of the staff of the Food Technology Department of the Technical University of Madrid, Spain. Assessments took place in standard sensory analysis chambers with separate booths. Following the generation of a consistent terminology by consensus, one visual descriptor, four aromas and four taste attributes were chosen to describe the wines. Formal assessment consisted of two sessions held on different days where wine tasters tasted all fermented triplicates. The panellists used a 10-cm unstructured scale, from 0 (no character) to 10 (very strong character), to rate the intensity of the 10 attributes.

Statistical analyses

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

Results and Discussion

First phase of S. pombe strain selection

A specific advantage of Schizosaccharomyces yeast in winemaking is that it degrades malic acid. To examine this with our strains, we initiated fermentation of grape must supplemented with malic acid (4 g/L). After fermenting the 75 S. pombe strains in test tubes for 21 days, we measured malic acid. We also measured two basic parameters, residual glucose + fructose and acetic acid production, which are typically used in the selection of fermentative yeasts of the genus Saccharomyces, the main yeast genus used in winemaking. The final concentrations of glucose + fructose after fermentation were close to 0 g/L (Fig 1a). This finding is in agreement with data reported by other authors, and is indicative of strong fermentative power [37,3]. This property allows the production of dry wines that are most popular in the world market.

Fig 1. (a) Glucose + fructose concentrations (g/L); (b) malic acid degradation (%); (c) acetic acid concentration (g/L).

Fig 1

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Scatterplots and box-and-whisker plots for the mean values of the final fermentations for the 75 studied S. pombe strains after 21. The red points indicate the values of the standard laboratory reference strain (Leupolds, 972, 975, 978), green line indicates values of strains with low levels of acetic acid (<0.4 g/L).

The degradation of malic acid, which currently represents the primary industrial application of Schizosaccharomyces yeasts, was nearly 100% in all the studied strains (Fig 1b). These data are comparable to those from other authors, who reported malic acid degradation rates ranging between 75 and 100% [38].

Out of the 75 initial S. pombe strains tested, however, only five strains (JB4/NCYC683, JB837/UWOPS92.229.4, JB873/NCYC3422, JB899/Y470 and JB917/CBS1057) showed moderate final concentrations of acetic acid (<0.4 g/L) (Fig 1c). According to these results, just 6.5% of the S. pombe strains could be used for producing quality wine. In a previous study, a similarly low rate of 5% was reported [5]. Three of these strains (JB837, JB873 and JB899) showed acetic acid concentration lower than 0.3 g/L. These data demonstrate that it is possible to identify S. pombe strains that can produce quality wines (Fig 1c).

Comparison between pre-selected S. pombe strains and other culture collection strains

After identifying the three S. pombe strains that show low acetic acid production, we studied additional features in detail. Microvinifications (200 mL) were performed to verify the acetic acid data and to confirm the proper fermentative ability of the pre-selected strains. In this portion of the study, the pre-selected S. pombe strains (JB837, JB873 and JB899) were compared with two S. cerevisiae strains (87/CECT12512 and 88/CECT12513) and with three other S. pombe strains (V1, 935/CECT1376 and 936/CECT12774) that had been tested previously [5].

Acetic acid

The maximal final concentrations of acetic acid were approximately 0.3 g/L (Fig 2.a.1; Table 2) for all selected strains, while the S. pombe strains from the Spanish Type Culture Collection produced acetic acid concentrations of up to 1 g/L (Fig 2.b.1; Table 2). Therefore, when used as a single strain during fermentation, the latter strains are not suitable for the production of quality wines [14,5].

Fig 2. Fermentation kinetics of acetic acid (1), malic acid (2) and glucose + fructose (3) for (a) selected S. pombe strains (JB899/Y470, JB917/CBS1057, JB873/NCYC3422 and V1); (b) non-selected S. pombe strains from the collection (935 and 936); (c) selected S. cerevisiae strains (87 and 88).

Fig 2

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Malic acid

The malic acid degradation was almost complete for most of the studied S. pombe strains (Fig 2.a.2 and 2.b.2; Table 2), although the degradation kinetics differed. The two S. cerevisiae strains degraded 11–24% of the initial malic acid content in the must (Fig 2.c.2; Table 2). Several authors have proposed similarly high malic acid degradation for yeast belonging to other genera than Schizosaccharomyces [5, 39–43]. The malic acid reduction influenced the final pH of the wine (Table 2), as S. pombe fermentations showed up to 0.34 higher pH values than S. cerevisiae fermentations.

Sugar consumption kinetics

The consumption kinetics of glucose + fructose was more rapid in the S. cerevisiae strains than in most studied S. pombe strains (Fig 2.a.3, 2.b.3 and 2.c.3; Table 2). Similar results have been described before [6]. Nevertheless, differences in degradation kinetics of glucose + fructose between the studied S. pombe strains were evident (Fig 2.a.3 and 2.b.3).

Pyruvic acid

All studied S. pombe strains produced more pyruvic acid than the S. cerevisiae yeasts (Fig 3). Strain JB873 yielded a maximum pyruvic acid concentration of 487 mg/L after 48 h of fermentation, which is a slightly higher concentration than those obtained in previous studies [5,6]. The other S. pombe strains provided a maximum pyruvic acid content that varied from 254 to 276 mg/L, except for the case of S. pombe JB899 that reached a pyruvic acid content of 374 mg/L (Fig 3). Previous pyruvic acid studies with S. cerevisiae and non-Saccharomyces species other than Schizosaccharomyces showed maximum values of about 150 mg/L during the first stages of fermentation [43]. These values are substantially lower than those obtained for the studied S. pombe strains. There is a strong correlation between the amount of pyruvic acid released by yeasts and the formation of vitisin A [3,5,15]; vitisin A is a stable pigment that influences wine colour quality. Thus, the selected strains with high pyruvate production rates such as JB873 and JB899 may be of interest in red wine production.

Fig 3. Fermentation kinetics of pyruvic acid for selected S. pombe strains (JB899/Y470, JB917/CBS1057, JB873/NCYC3422 and V1), non-selected S. pombe strains from the collection (935 and 936) and selected S. cerevisiae strains (87 and 88).

Fig 3

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Glycerol

Schizosaccharomyces species have previously been reported to produce more glycerol than Saccharomyces species [15]. In this study, final levels of glycerol varied from 8.02 g/L to 8.91 g/L (Table 2). Even though two S. pombe strains showed the highest levels (V1 and JB917), several differences were observed between the studied strains, and not in every case were the values higher than those obtained for the two S. cerevisiae strains. Increased glycerol content has been described as one of the main contributors of non-Saccharomyces strains on wine quality [1,44].

Ethanol

Among all strains tested, the final ethanol levels varied from 12.04 to 12.44 (% vol/vol) (Table 2). The fermentations involving S. pombe produced very similar ethanol levels to the S. cerevisiae strains we examined (Table 2). Among the S. pombe strains, only slight differences in final ethanol levels of less than 0.24 (% vol/vol) were evident. In contrast other authors have shown that some non-Saccharomyces types of yeast produce lower ethanol yields than Saccharomyces [45–47]. The sugar metabolism can also be used to produce compounds other than ethanol, such as glycerol or pyruvic acid, or to increase the yeast biomass [48,49]. Previous studies with S. pombe showed similar results to those obtained here [13]. Other authors observed lower final ethanol levels using other non-Saccharomyces species under specific high aeration conditions [50,51].

Urea

The urea content of the finished wines was lower in the fermentations involving S. pombe, with values less than 0.5 mg/L (Table 2). This result can be attributed to the special ability of the Schizosaccharomyces genus to produce urease [12,52]. This enzymatic activity also could reduce the initial level of precursors for ethyl carbamate (one of the most toxic compounds reported in wine) [3,15]. This factor is becoming increasingly important as ethyl carbamate is a known carcinogen that is present in a variety of fermented foods [53].

Citric acid and lactic acid

No differences were evident with respect to citric and lactic acid (Table 2), as no wine performed malolactic fermentation [6] or Lachancea thermotolerans species were used [54]. Note that after a traditional malolactic fermentation process by lactic bacteria, all citric acid could be converted into acetic acid; this collateral effect increases the final acetic acid content [6] and consequently slightly reduces wine quality.

Volatile aromas

Higher alcohols were produced in higher total concentrations by S. cerevisiae fermentations (Table 3). Some differences were observed between the studied S. pombe strains (Table 3). Other authors described non-Saccharomyces yeasts as lower producers of higher alcohols than S. cerevisiae [42,43,54–57], and much strain variability has been reported [58,59]. This finding could be of interest in facilitating the making of wines with typicity for specific grape varieties or to increase wine complexity [60]. Similarly, the tested S. pombe strains produced less esters than the S. cerevisiae strains. No differences between the S. pombe and S. cerevisiae strains were observed with respect to compounds considered negative, such as ethyl acetate and diacetyl. However, those compounds could increase after a malolactic fermentation process performed by lactic bacteria [15]. Significant differences in compounds such as acetaldehyde and ethyl acetate were evident between the different S. pombe strains.

Biogenic amines

During the past years, harmful effects of biogenic amines [61–64] have been demonstrated, which now constitutes a serious matter in food safety that must be taken into account. A histamine value of 2 mg/L is considered the maximum level [65] in some countries. All studied strains reported values below that threshold (Table 4). The study shows that S. pombe does not produce higher levels of biogenic amines than S. cerevisiae. Subtle statistical differences were observed in the case of cadaverine (Table 4), but this biogenic amine is not produced by yeasts, and the slight differences could reflect that some strains remove biogenic amines during the fermentation process; similar processes have been described before [66]. However, most biogenic amines are produced during wine ageing and malolactic fermentation [67], as they are compounds produced primarily by lactic acid bacteria [68,69]. Thus, the wines that still contain malic acid (Fermentations by S. cerevisiae) could increase their biogenic amine contents later on [6,16].

Amino acids

S. pombe fermentations showed higher final levels in most amino acids (Table 5). Similar results were obtained before, as it has been described as demanding less nitrogen [14] and releasing more nitrogen [7] than S. cerevisiae. However some differences were observed among the studied strains (Table 5). S. cerevisiae fermentations produced a higher final level in ornithine. The observed differences in threonine, valine, isoleucine and leucine explain the differences measured for higher alcohols (Table 5), because they are precursors of 1-propanol, isobutanol, 2-methylbutanol and 3-methylbutanol, respectively (Table 5) [43]. The statistical differences reported for histidine, tyrosine and lysine show that S. pombe fermentations increase the content of some biogenic amine precursors [65,67]. Nevertheless, the transformation of some of those precursors to biogenic amines takes place during the malolactic fermentation, therefore the wines fermented by S. pombe did not run a serious risk of increasing the levels of histamine or tyramine since they do not need any longer a malolactic fermentation [16].

Fermentations in stress conditions

Several authors have reported how important is to study yeast performance under stress conditions [70,71]. After performing the stress tests conditions for the selected strains (Table 6) for some parameters described before in literature [34]. We observed that different phenotypic cases were detected for the studied strains in the studied parameters. Other authors have reported before the importance of phenotypic performance in yeasts [72,73]. According to these results strain S.pombe 936/CECT12774 would develop better than the others S.pombe under circumstances similar to KCl test and strain JB837/NOTT1 would not be able to perform properly. In the NaCl test strains JB873/NCYC3422, JB4/NCYC683, 935/CECT1376 and 936/CECT12774 performed slightly better than the others S.pombe. For the ethanol resistance tests, strains JB873/NCYC3422, 935/CECT1376 and 936/CECT12774 could perform better than the others. In the case of KHSO3 test, strains JB873/NCYC3422, JB4/NCYC683, 935/CECT1376 and 936/CECT12774 showed to be more resistant. The highest fermenting powers were obtained by strains JB873/NCYC3422, JB4/NCYC683, 935/CECT1376 and 936/CECT12774 among the S.pombe strains. Even though the values were better for the S.cerevisiae controls, these differences could be related to the slower developing kinetic describe for S.pombe than S.cerevisiae that was observed in the previous trials and reported by other authors [6]. In fact S.pombe has been described before as highly resistant to some stress conditions [23].

Table 6. Final analysis of phenotypic classes for different stressful parameters.

Fermentations by selected S. pombe strains (JB899/Y470, JB917/CBS1057, JB873/NCYC3422, JB4/ NCYC683, JB837/NOTT1 and V1), non-selected S. pombe strains from the Spanish culture collection (936/CECT12774 and IFI 935/CECT1376) and selected S. cerevisiae strains (IFI 87/CECT12512 and IFI 88/CECT12513). Number of strains belonging to different phenotypic classes, regarding values of optical density after developing in corrected must with studied stressful parameter (Class 0: A640 = 0.1; Class 1: 0.2<A640>0.4; Class 2: 0.5<A640>1.0; Class 3: A640>1.0).

Compounds (mg/L) 899 917 873 4 837 V1 935 936 87 88
KCl (0.75 M) 1 1 2 2 0 1 2 3 3 3
NaCl (1.5 M) 0 0 1 1 0 0 1 1 1 1
Ethanol 6% (% v/v) 1 1 2 1 1 1 1 2 3 2
Ethanol 10% (% v/v) 0 0 1 0 0 0 1 1 1 1
Ethanol 12% (% v/v) 0 0 0 0 0 0 0 0 1 1
Ethanol 14% (% v/v) 0 0 0 0 0 0 0 0 0 0
KHSO3 (150 mg/L) 2 2 3 3 1 2 3 3 3 3
KHSO3 (300 mg/L) 1 1 2 2 0 1 2 2 3 3
Fermenting power (Ethanol (% v/v)) 11.21±0.18b 11.39±0.24b 12.36±0.21c 12.42±0.23c 10.68±0.28a 11.33±0.22b 12.44±0.31c 12.61±26c 13.81±0.29d 13.68±0.33d
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Results represent the mean ± SD for three replicates. Means in the same row with the same letter are not significantly different (p < 0.05).

Sensory evaluation

A sensory evaluation was performed to verify that the selected strains had the potential to produce wines that were pleasant to drink. Fig 4 shows a spider web diagram of the average scores of some olfactory and taste attributes. Large differences in the perception of acidity were recorded; this result agrees with acidity parameters explained above in the previous fermentations. Slight differences were reported regarding to sweetness even though all fermentations depleted successfully all the sugars, this can be explained by differences in acid-sweet balance. Serious faults were reported for non-selected S. pombe strains IFI935/CECT1376 and 936/CECT12774 regarding to high acetic acid and reduction characters. Fermentations by selected S. pombe strains JB899/Y470 and JB873/NCYC3422 received the best scores from all tasters while non-selected IFI935/CECT1376 and 936/CECT12774 received the lowest scores, S. cerevisiae strains IFI87/CECT12512 and IFI88/CECT12513 received moderate scores in overall impression scores related to excessive high acidity for the tasters. These results show that S. pombe can perform better than S.cerevisiae under very acidic conditions. The above data show that all fermentations with S. pombe achieved the main goals related to malic acid deacidification from a very acidic must.

Fig 4. Results of the sensory analysis on fermentation products of selected S. pombe strains: JB899/Y470, JB873/NCYC3422, JB917/CBS1057, JB4/NCYC683, JB837/NOTT1 and V1; Non selected S.pombe strains: 936/CECT12774 and IFI935/CECT1376; Selected S. cerevisiae strains: IFI87/CECT12512 and IFI88/CECT12513.

Fig 4

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Conclusions

Among 75 S. pombe strains tested, we have discovered three strains with highly promising winemaking properties. These strains could be used to produce wines with low levels of malic acid, acetic acid, ethyl carbamate and biogenic amines, and with an appropriate volatile aroma profile. Because these S. pombe strains produced similar ethanol as the budding yeast strains we examined, this species may well have untapped potential for the producing of ethanol as a fuel (‘bioethanol’).

Supporting Information

S1 Table. First Selection Phase Results.

(XLSX)

Click here for additional data file. (30.1KB, xlsx)

Acknowledgments

The authors are grateful to the accredited Estación Enológica de Haro laboratory, where the biogenic amines, amino acids and volatile aroma analysis were performed, especially to Montserrat Iñiguez and Elena Melendez. Jürg Bähler and Daniel Jeffares were supported by a Wellcome Trust Senior Investigator Award to J.B. (grant 095598/Z/11/Z).

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

Jürg Bähler and Daniel Jeffares were supported by a Wellcome Trust Senior Investigator Award to JB, Grant Number: 095598/Z/11/Z, URL: http://www.wellcome.ac.uk/. The rest of the authors have no support or funding to report. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Su J, Wang T, Wang Y, Li YY, Li H. The use of lactic acid-producing, malic acid-producing, or malic acid-degrading yeast strains for acidity adjustment in the wine industry. Appl Microbiol Biotechnol. 2014; 98: 2395–2413. 10.1007/s00253-014-5508-y [DOI] [PubMed] [Google Scholar]
  • 2.Jolly NP, Varela C, Pretorius IS. Not your ordinary yeast: Non-Saccharomyces yeasts in wine production uncovered. FEMS Yeast Res. 2014; 14: 215–237. 10.1111/1567-1364.12111 [DOI] [PubMed] [Google Scholar]
  • 3.Benito S, Palomero P, Calderón F, Palmero D, Suárez-Lepe JA. Schizosaccharomyces In: Batt CA, Tortorello ML, editors. Encyclopedia of Food Microbiology (2nd ed.), VOL. 3: Elsevier, Amsterdam, the Netherlands; 2014. pp. 365–370. [Google Scholar]
  • 4.Ding S, Zhang Y, Zhang J, Zeng W, Yang Y, Guan J, et al. Enhanced deacidification activity in Schizosaccharomyces pombe by genome shuffling. 2015; 32(2): 317–25. [DOI] [PubMed] [Google Scholar]
  • 5.Benito S, Palomero P, Calderón F, Palmero D, Suárez-Lépe JA. Selection of appropriate Schizosaccharomyces strains for winemaking. Food Microbiol. 2014; 42: 218–224. 10.1016/j.fm.2014.03.014 [DOI] [PubMed] [Google Scholar]
  • 6.Benito A, Palomero F, Calderón F, Benito S. Combine Use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans Yeast Strains as an Alternative to the Traditional Malolactic Fermentation in Red Wine Production. Molecules. 2015a; 20: 9510–9523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Palomero F, Morata A, Benito S, Calderón F, Suárez-Lepe JA. New genera of yeasts for over-lees aging of red wine. Food Chem. 2009; 112: 432–441. [Google Scholar]
  • 8.Peinado RA, Moreno JJ, Maestre O, Ortega JM, Medina M, Mauricio JC. Gluconic acid consumption in wines by Schizosaccharomyces pombe and its effect on the concentrations of major volatile compounds and polyols. J Agric Food Chem. 2004a; 52: 493–497. [DOI] [PubMed] [Google Scholar]
  • 9.Peinado RA, Mauricio JC, Medina M, Moreno JJ. Effect of Schizosaccharomyces pombe on aromatic compounds in dry sherry wines containing high levels of gluconic acid. J Agric Food Chem. 2004b; 52: 4529–4534. [DOI] [PubMed] [Google Scholar]
  • 10.Peinado RA, Moreno JJ, Medina M, Mauricio JC. Potential application of a glucose-transport-deficient mutant of Schizosaccharomyces pombe for removing gluconic acid from grape must. J. Agric. Food Chem. 2005; 53, 1017–1021. [DOI] [PubMed] [Google Scholar]
  • 11.Peinado RA, Moreno JJ, Maestre O. Removing gluconic acid by using different treatments with a Schizosaccharomyces pombe mutant: effect on fermentation by products. Food Chem. 2007; 104: 457–465. [Google Scholar]
  • 12.Deák T. Handbook of Food Spoilage Yeasts. 2nd ed CRC Press: Taylor & Francis Group, Boca Raton, USA; 2008. pp. 294–297. [Google Scholar]
  • 13.Benito S, Palomero F, Morata A, Calderon F, Palmero D, Suarez-Lepe JA. Physiological features of Schizosaccharomyces pombe of interest in making of white wines. Eur Food Res Technol. 2013b; 236: 29–36. [Google Scholar]
  • 14.Benito S, Palomero P, Morata A, Calderón F, Suárez-Lépe JA. New applications for Schizosaccharomyces pombe in the alcoholic fermentation of red wines. Int J Food Microbiol. 2012; 47: 2101–2108. [DOI] [PubMed] [Google Scholar]
  • 15.Benito S, Palomero P, Gálvez L, Morata A, Calderón F, Palmero D, et al. Quality and Composition of Red Wine Fermented with Schizosaccharomyces pombe as Sole Fermentative Yeast, and in Mixed and Sequential Fermentations with Saccharomyces cerevisiae. Food Technol Biotechnol. 2014c; 52: 376–382. [Google Scholar]
  • 16.Benito S. The use of Schizosaccharomyces yeast in order to reduce the content of biogenic amines and ethyl carbamate in wines. J Food Process Technol. 2015c; 6(9): 9–10. [Google Scholar]
  • 17.Gallander JF. Deacidification of eastern table wines with Schizosaccharomyces pombe. Am J Enol Vitic. 1977; 28: 65–72. [Google Scholar]
  • 18.Snow PG, Gallander JF. Deacidification of white table wines trough partial fermentation with Schizosaccharomyces pombe. A J Enol Vitic. 1979; 30: 45–48. [Google Scholar]
  • 19.Unterholzner O, Aurich M, Platter K, Geschmacks und Geruchsfehler bei Rotweinen verursacht durch Schizosaccharomyces pombe L. Mitteilungen Klosterneuburg, Rebe und Wein, Obstbau und Früchteverwertung. 1988; 38: 66–70. [Google Scholar]
  • 20.Yokotsuka K, Otaki A, Naitoh H. Controlled Simultaneous deacidification and alcohol fermentation of a high acid grape must using two immobilized yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae. A. J. Enol. Vit. 1993; 44: 371–377. [Google Scholar]
  • 21.Pitt JI, Hocking AD. Fungi and Food Spoilage. 2nd ed An Aspen Publication: Gaithersburg; 1999. pp. 459–460. [Google Scholar]
  • 22.http://www.proenol.com/files/editorials/ProMalic_FT204-09_PT.pdf?u=. Accessed 18 September 2015.
  • 23.Benito S, Gálvez L, Palomero F, Calderón F, Morata A, Suárez-Lepe JA. Schizosaccharomyces selective differential media. Afr. J. Microbiol. Res. 2013; 7: 3026–3036. [Google Scholar]
  • 24.Jeffares DC, Rallis C, Rieux A, Speed A, Převorovský M, Mourier T, et al. The genomic and phenotypic diversity of Schizosaccharomyces pombe. Nat Genet. 2015; 47: 235–241. 10.1038/ng.3215 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Bai FY. State Key Laboratory of Mycology, http://www.mycolab.org.cn/templates/T_second_EN/index.aspx?nodeid=430. Accessed 18 September 2015.
  • 26.Pretorius IS, Bauer FF, Meeting the consumer challenge through genetically customized wine-yeast strains. Trends in Biotechnol. 2002; 20: 426–432. [DOI] [PubMed] [Google Scholar]
  • 27.Husnik JL, Volschenk H, Bauer J. Metabolic engineering of malolactic wine yeast. Metabol Eng. 2006: 8: 315–323. [DOI] [PubMed] [Google Scholar]
  • 28.Husnik JL, Delaquis PJ, Cliff MA. Am J Enol Vitic. 2007; 58:42–52 [Google Scholar]
  • 29.Liu YL, Li H. Integrated Expression of the Oenococcus oeni mleA Gene in Saccharomyces cerevisiae. Agr Sci China. 2009; 8: 821–827. [Google Scholar]
  • 30.http://www.magrama.gob.es/es/calidad-y-evaluacion-ambiental/temas/biotecnologia/organismos-modificados-geneticamente-omg-/legislacion-general/legislacion_europea.aspx. Accessed 18 September 2015.
  • 31.Vaughnan-Martini A, Martini A. Determination of ethanol production In: Kurtzman CP, Fell JW, editors. The Yeast. A Taxonomic Study. Elsevier: Amsterdam, Netherlands; 1999. p. 107. [Google Scholar]
  • 32.http://www.biosystems.es/products/ENOLOGY/Reactivos_EnologY/ENZIM%c3%81TICOS. Accessed 18 September 2015.
  • 33.http://shop.gabsystem.com/b2c/producto/1010004/1/ebulliometro-gab. Accessed 18 September 2015.
  • 34.Mendes I, Franco-Duarte R, Umek L, Fonseca E, Drumonde-Neves J, Dequin S, et al. Computational Models for Prediction of Yeast Strain Potential for Winemaking from Phenotypic Profiles. PLOS One.2013, 8(7): 1–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Vaughan-Martini A, Martín A. Determination of ethanol production In: The yeasts. A taxonomic study. Eds. Kurtzman CP, Fell JW, Elsevier; 1998; p 107. [Google Scholar]
  • 36.http://www.oiv.int/oiv/files/3%20-%20Resolutions/EN/2012/OIV-OENO%20370-2012.pdf.
  • 37.Fleet GH. Wine yeasts for the future. FEMS Yeast Res. 2008; 8(7): 979–995. 10.1111/j.1567-1364.2008.00427.x [DOI] [PubMed] [Google Scholar]
  • 38.Silva S, Ramón-Portugal F, Andrade P, Texera M, Strehaino P. Malic acid consumption by dry immobilized cells of Schizosaccharomyces pombe. A J Enol Vitic. 2003; 54: 50–55. [Google Scholar]
  • 39.Rodriguez SB, Thornton RJ. Factors influencing the utilisation of L-malate by yeasts. FEMS Microbiol Lett. 1990; 72: 17–22. [DOI] [PubMed] [Google Scholar]
  • 40.Thornton RJ, Rodriguez SB. Deacidification of red and white wines by a mutant of Schizosaccharomyces malidevorans under commercial winemaking conditions. Food Microbiol. 1996; 13: 475–482. [Google Scholar]
  • 41.Redzepovic S, Orlic S, Majdak A, Kozima B, Volschenk H, Viljoen-Bloom M. Differential malic acid degradation by selected strains of Saccharomyces during alcoholic fermentation. Int J Food Microbiol. 2003; 83: 49–61. [DOI] [PubMed] [Google Scholar]
  • 42.Belda I, Navascués E, Marquina D, Santos A, Calderón F, Benito S. Dynamic analysis of physiological properties of Torulaspora delbrueckii in wine fermentations and its incidence on wine quality. Appl Microbiol Biotechnol. 2015; 99(4): 1911–22. 10.1007/s00253-014-6197-2 [DOI] [PubMed] [Google Scholar]
  • 43.Benito S, Hofmann T, Laier M, Lochbühler BC, Schüttler A, Ebert K, et al. Effect on quality and composition of Riesling wines fermented by sequential inoculation with non-Saccharomyces and Saccharomyces cerevisiae. Eur Food Res Technol. 2015b; 241(5):707–717. [Google Scholar]
  • 44.Jolly NP, Augustyn OPH, Pretorius IS. The Role and Use of Non-Saccharomyces Yeasts in Wine Production. S Afr J Enol Vitic. 2006; 27: 15–39. [Google Scholar]
  • 45.Kutyna DR, Varela C, Henschke PA, Chambers PJ, Stanley GA. Microbiological approaches to lowering ethanol concentration in wine. Trends Food Sci. Technol. 2010; 21: 293–302. [Google Scholar]
  • 46.Gobbi M, Comitini F, Domizio P, Romani C, Lencioni L, Mannazzu I, et al. Fermentative aptitude of non-Saccharomyces wine yeast for reduction in the ethanol content in wine. Eur Food Res Technol. 2014; 239(1): 41–48. [Google Scholar]
  • 47.Contreras A, Hidalgo C, Henschke PA, Chambers PJ, Curtin C, Varela C. Evaluation of Non-Saccharomyces Yeasts for the Reduction of Alcohol Content in Wine. Appl Environ Microb. 2014; 80: 1670–1678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Merico A, Sulo P, Piskur J, Compagno C. Fermentative lifestyle in yeasts belonging to the Saccharomyces complex. FEBS J. 2007; 274: 976–989. [DOI] [PubMed] [Google Scholar]
  • 49.Bely M, Stoeckle P, Masneuf-Pomarède I, Dubourdieu D. Impact of mixed Torulaspora delbrueckii–Saccharomyces cerevisiae culture on high-sugar fermentation. Int J Food Microbiol. 2008; 122: 312–320. 10.1016/j.ijfoodmicro.2007.12.023 [DOI] [PubMed] [Google Scholar]
  • 50.Contreras A, Curtin C, Varela C. Yeast population dynamics reveal a potential 'collaboration' between Metschnikowia pulcherrima and Saccharomyces uvarum for the production of reduced alcohol wines during Shiraz fermentation. Appl Microbiol Biotechnol. 2015; 99(4): 1885–95. 10.1007/s00253-014-6193-6 [DOI] [PubMed] [Google Scholar]
  • 51.Morales P, Rojas V, Quirós M, González R. The impact of oxygen on the final alcohol content of wine fermented by a mixed starter culture. Appl Microbiol Biotechnol. 2015; 99(9): 3993–4003. 10.1007/s00253-014-6321-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Lubbers MW, Rodriguez SB, Honey NK, Thornton RJ. Purification and characterization of urease from Schizosaccharomyces pombe. Canad J Microbiol. 1996; 42: 132–40. [DOI] [PubMed] [Google Scholar]
  • 53.Xia C, Hong M, YunFeng Z, Ning WY. Research progress on toxicity and contamination of ethyl carbamate in fermented foods. J Food Safety Quality. 2014; 5(9): 2617–262. [Google Scholar]
  • 54.Benito A, Calderón F, Palomero F, Benito S. Quality and Composition of Airen Wines Fermented by Sequential Inoculation of Lachancea thermotolerans and Saccharomyces cerevisiae. Food Technol. Biotechnol. Accepted 2015. 10.17113/ftb.54.02.16.4220 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Clemente-Jiménez JF, Mingorance-Cazorla L, Martínez-Rodríguez S, Las Heras-Vázquez FJ, Rodríguez-Vico F. Molecular characterization and oenological properties of wine yeast isolated during spontaneous fermentation of six varieties of grape must. Food Microbiol. 2004; 21: 149–155. [Google Scholar]
  • 56.Parapouli M, Hatziloukas E, Drainas C, Perisynakis A. The effect of Debina grapevine indigenous yeast strains of Metschnikowia and Saccharomyces on wine flavour. J Ind Microbiol Biot. 2010; 37: 85–93. [DOI] [PubMed] [Google Scholar]
  • 57.Gobbi M, Comitini F, Domizio P, Romani C, Lencioni L, Mannazzu I, et al. Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine. Food Microbiol. 2013; 33: 271–281. 10.1016/j.fm.2012.10.004 [DOI] [PubMed] [Google Scholar]
  • 58.Romano P, Suzzi G, Comi G, Zironi R. Higher alcohol and acetic acid production by apiculate wine yeasts. J Appl Bact. 1992; 73: 126–130. [Google Scholar]
  • 59.Zironi R, Romano P, Suzzi G, Battistutta F. Comi G. Volatile metabolites produced in wine by mixed and sequential cultures of Hanseniaspora guilliermondii or Kloeckera apiculata and Saccharomyces cerevisiae. Biotechnol Lett. 1993; 15: 235–238. [Google Scholar]
  • 60.Romano P, Suzzi G. Higher alcohol and acetoin production by Zygosaccharomyces wine yeasts. J Appl Bacteriol. 1993; 75: 541–545. [Google Scholar]
  • 61.Maynard LS, Schenker VJ. Monoamine-oxidase inhibition by ethanol in vitro. Nature. 1996; 196: 575–576. [DOI] [PubMed] [Google Scholar]
  • 62.Kanny G, Gerbaux V, Olszewski A, Frémont S, Empereur F, Nabet F, et al. No correlation between wine intolerance and histamine content of wine. J Allergy Clin Inmmun. 2001; 107: 375–378. [DOI] [PubMed] [Google Scholar]
  • 63.Jansen SC, van Dusseldorp M, Bottema KC, Dubois AE. Intolerance to dietary biogenic amines: A review. Ann Allerg Asthama Im. 2003; 91: 233–240. [DOI] [PubMed] [Google Scholar]
  • 64.Moreno-Arribas MV, Polo MC. Occurrence of lactic acid bacteria and biogenic amines in biologically aged wines. Food Microbiol. 2008; 25: 875–881. 10.1016/j.fm.2008.05.004 [DOI] [PubMed] [Google Scholar]
  • 65.Lehtonen P. Determination of amines and amino acids in wine: A review. Am J Enol Vitic. 1996; 47(2): 127–133. [Google Scholar]
  • 66.Medina K, Boido E, Fariña L, Gioia O, Gomez ME, Barquet M, et al. Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae. Food Chem. 2013, 141(3):2513–21. 10.1016/j.foodchem.2013.04.056 [DOI] [PubMed] [Google Scholar]
  • 67.Alcaide-Hidalgo JM, Moreno-Arribas MV, Martín-Álvarez PJ, Polo MC. Influence of malolactic fermentation, postfermentative treatments and ageing with lees on nitrogen compounds of red wines. Food Chem. 2007; 103: 572–581. [DOI] [PubMed] [Google Scholar]
  • 68.Smit AY, du Toit M. Evaluating the Influence of Malolactic Fermentation Inoculation Practices and Ageing on Lees on Biogenic Amine Production in Wine. Food Bioprocess Technol. 2013; 6: 198–206. [Google Scholar]
  • 69.Sumby KM, Grbin PR, Jiranek V. Implications of new research and technologies for malolactic fermentation in wine. Appl Microbiol Biotechnol. 2014; 98: 8111–8132. 10.1007/s00253-014-5976-0 [DOI] [PubMed] [Google Scholar]
  • 70.Zheng YL, Wang SA. Stress Tolerance Variations in Saccharomyces cerevisiae Strains from Diverse Ecological Sources and Geographical Locations. PLOS One. 2015. 10.1371/journal.pone.0133889 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Bonciani T, Solieri L, De Vero L, Giudici P. Improved wine yeasts by direct mating and selection under stressful fermentative conditions. Eur Food Res Technol. 2015, Accepted 10.1007/s00217-015-2596-6 [DOI] [Google Scholar]
  • 72.Camarasa C, Sanchez I, Brial P, Bigey F, Dequin S. Phenotypic Landscape of Saccharomyces cerevisiae during Wine Fermentation: Evidence of Origin-Dependent Metabolic Traits. PLOS One.2011, 6(9): 1–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Hyma KE, Saerens SM, Verstrepen KJ, Fay JC. Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae. FEMS Yeast Res. 2011, 11(7):540–51. 10.1111/j.1567-1364.2011.00746.x [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

S1 Table. First Selection Phase Results.

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