New applications for Schizosaccharomyces pombe in the alcoholic fermentation of red wines

Original article

New applications for Schizosaccharomyces pombe in the alcoholic

fermentation of red wines

Santiago Benito,* Felipe Palomero, Antonio Morata, Fernando Calderon & Jose A. Suarez-Lepe

Depto. de Tecnologıa de Alimentos, Escuela Tecnica Superior de Ingenieros Agronomos, Universidad Politecnica de Madrid, Ciudad Universitaria

S ⁄N, 28040 Madrid, Spain

(Received 28 March 2012; Accepted in revised form 2 April 2012)

Summary The fermentation of grape must using non-Saccharomyces yeasts with particular metabolic and biochemical

properties is of growing interest. In the present work, red grape must was fermented using four strains of

Schizosaccharomyces pombe (935, 936, 938 and 2139), Saccharomyces cerevisiae 7VA and Saccharomyces

uvarum S6U, and comparisons were made over the fermentation period in terms of must sugar

(glucose + fructose), malic acid, acetic acid, ammonia, primary amino nitrogen, lactic acid, urea (a

possible fermentation activator or precursor of other metabolites) and pyruvic acid (a molecule affecting

vitisin formation and therefore colour stability) concentration. The colour intensity of the fermenting musts

was also recorded. The Schizosaccharomyces strains consumed less primary amino nitrogen and produced

less urea and more pyruvic acid than other Saccharomyces species. Further, three of the four

Schizosaccharomyces strains completed the breakdown of malic acid by day 4 of fermentation. The main

negative effect of the use of Schizosaccharomyces was strong acetic acid production. The Schizosacchar-

omyces strains that produced most pyruvic acid (938 and 936) were associated with better ‘wine’ colour than

the remaining yeasts. The studied Schizosaccharomyces could therefore be of oenological interest.

Keywords Malic acid, pyruvic acid, Saccharomyces spp., Schizosaccharomyces pombe, urea.

Introduction

The yeasts of the genus Schizosaccharomyces havetraditionally been described as wine spoilage organismsowing to their production of compounds with negativesensorial impacts, such as acetaldehyde, H2S and volatileacids (Gallander, 1977; Snow & Gallander, 1979; Unter-holzner et al., 1988; Yokotsuka et al., 1993; Pitt &Hocking, 1999). However, the industrial use of Schizo-saccharomyces has been described in the fermentation ofcane sugar in rum-making (Pech et al., 1984; Fahrasmaneet al., 1988), the production of palm wine (Christopher &Theivendirarajah, 1988; Sanni & Loenner, 1993) andcocoa fermentation (Ravelomanana et al., 1984;Mazigh,1994). The genus has also been studied at the laboratoryand semi-industrial scales in the winemaking industrygiven the notable capacity of some of its members todeacidify wines via the ability to metabolise malic acidwith the production of ethanol (Gallander, 1977; Snow &Gallander, 1979; Sousa et al., 1993, 1995; Yokotsuka

et al., 1993; Gao & Fleet, 1995; Thornton & Rodrıguez,1996; Dharmadhikari & Wilker, 1998). In northerlyviticultural regions, where grape malic acid contents canbe high, the possible use of non-Saccharomyces yeasts,such as Schizosaccharomyces spp., to reduce malic acidconcentrations is awakening much interest (Gallander,1977;Magyar &Panik, 1989; Seo et al., 2007; Fleet, 2008;Kim et al., 2008; Kunicka-Styczynska, 2009). Recently,the OIV approved ‘deacidification by Schizosaccharomy-ces, (Resolution OENO ⁄MICRO ⁄97 ⁄75 ⁄phase 7), butthe number of commercial strains available for this is verylimited. Mixed and sequential cultures with Saccharomy-ces (Kim et al., 2008; Kunicka-Styczynska, 2009) havebeen used to mitigate the negative effects of the currentlyavailable Schizosaccharomyces strains’ scant oenologicalaptitude (Unterholzner et al., 1988). Understanding howto isolate and select more appropriate Schizosacchar-omyces strains is therefore of great interest.One of the new applications of Schizosaccharomyces is

ageing over lees, made possible by these yeasts’ strongautolytic release of cell wall polysaccharides (Palomeroet al., 2009). Further, certain Schizosaccharomycesmutants may be able to reduce the gluconic acid

*Correspondent: Fax: +34 91 336 57 46;

e-mail: [email protected]

International Journal of Food Science and Technology 2012 1

doi:10.1111/j.1365-2621.2012.03076.x

� 2012 The Authors. International Journal of Food Science and Technology � 2012 Institute of Food Science and Technology

contents of spoiled musts (Peinado et al., 2007, 2009).The urease activity of Schizosaccharomyces spp. (Casas,1999; Barnett et al., 2000; Deak, 2008) is also of interestwith respect to food safety; its production could reducehigh wine ethyl carbamate contents by reducing ureaconcentrations (a precursor of ethyl carbamate)(Uthurry et al., 2004).To further our knowledge of the fermentative activity

of Schizosaccharomyces, the present work examined thefermentation kinetics of four strains of Schizosacchar-omyces pombe, along with the consumption of nitrogen-ated compounds and the production of acetic andpyruvic acids. The must colour changes that occurredover fermentation were also recorded.

Materials and methods

Yeast strains

The yeasts used in this study were Schizosaccharomycespombe strains 935, 936, 938 and 2139 from the typecollection of the Instituto de Fermentaciones Industri-ales (IFI, CSIC, Madrid, Spain), Saccharomyces cerevi-siae 7VA from the collection of the Departamento deTecnologıa de Alimentos de la Escuela Tecnica Superiorde Ingenieros Agronomos (Univeridad Politecnica,Madrid, Spain) and Saccharomyces uvarum S6U sup-plied by the Lallemand company (Danstar Ferment,Montreal, Canada).

Must preparation

All fermentations were performed using grape red muststock (224 g L)1 glucose + fructose [G + F]) from theRibera del Duero denomination of origin region (grapevariety Vitis vinifera cv. Syrah). The malic acid contentof the stock was adjusted to 2 g L)1 (Panreac, Barce-lona, Spain) via the addition of this compound (final pH3.5).

Fermentations

Microfermentations were performed using 50 mL ofmust inoculated with 1 mL of liquid YEDP mediumcontaining 108 cfu mL)1 (determined using a Thomaschamber) of one of the above-mentioned yeasts. Allfermentations were performed in 100-mL flasks sealedwith a Muller valve filled with 98% H2SO4 (Panreac);this allowed the release of CO2 while avoiding microbialcontamination (Vaughnan-Martini & Martini, 1999).The temperature was maintained at 25 �C. The fermen-tations proceeded without aeration, oxygen injection oragitation. All fermentations were performed in tripli-cate.The G + F, malic acid, acetic acid, ammonia,

primary amino nitrogen (PAN) and pyruvic acid con-

tents of the fermentations were monitored over a periodof 31 days. The colour intensity of the fermenting mustwas also recorded. The urea concentration was deter-mined at the end of fermentation.

Determination of glucose + fructose, malic acid, lactic acid,acetic acid, ammonia, primary amino nitrogen, urea,pyruvic acid and colour intensity

All analyses were undertaken using a Y15 Autoanalyzer(Biosystems, Barcelona, Spain). Enzymatic analyses forG + F, malic acid, lactic acid, acetic acid, ammonia,PAN and urea, and the colour intensity analysis, wereperformed using kits from Biosystems (http://www.biosystems.es). Pyruvic acid was determined using theappropriate kit (Megazyme, Bry, Ireland).

Statistical analysis

Means and standard deviations were calculated, andanova and least significant differences (LSD) tests wereperformed using PC Statgraphics v.5 software (GraphicsSoftware Systems, Rockville, MD, USA). Significancewas set at P < 0.05 for the anova matrix F-value. Themultiple range test was used to compare the means.

Results and discussion

Glucose + fructose fermentation

Figure 1 shows the fermentation kinetics of the differentyeasts examined. Differences can be seen between themembers of Saccharomyces and Schizosaccharomyces.Saccharomyces cerevisiae 7VA and S. uvarum S6Ufinished fermentation on days 4 and 11, respectively,although S6U left some residual sugar. Schizosacchar-omyces pombe 935, 936, 938 and 2139 required 15 daysto complete fermentation, leaving very little residualsugar. This result agrees with the high fermentativepower of this species reported by other authors (Pey-naud & Sudraud, 1962; Suarez-Lepe & Inigo, 2004). Theslower kinetics of Schizosaccharomyces would likelyallow the easier control of the temperature rises thatoccur over fermentation.

Degradation of malic acid

Schizosaccharomyces pombe 936, 938 and 2139 consumedall the malic acid present, while strain 935 reduced itspresence by 50% (Fig. 2). This result agrees with thatreported by other authors (75–100% depending on thestrain and culture medium) (Snow & Gallander, 1978;Magyar & Panik, 1989; Gao & Fleet, 1995; Taillandieret al., 1995; Thornton & Rodrıguez, 1996; Silva et al.,2003; De Fatima et al., 2007). Malic acid can be metab-olised by species other than Schizosaccharomyces

New Schizosaccharomyces pombe applications S. Benito et al.2

International Journal of Food Science and Technology 2012 � 2012 The Authors

International Journal of Food Science and Technology � 2012 Institute of Food Science and Technology

(Corte-Real et al., 1989; Corte-Real & Leao, 1990;Rodriguez&Thornton, 1990, 1990; Suarez-Lepe& Inigo,2004), although they reduce its presence by only some25%. The present Saccharomyces species also reduced themalic acid content, but this only amounted to 20% withS. cerevisiae 7VA and 30% with S. uvarum S6U.At the first control point (4 days), Schiz. pombe 936

and 938 had already consumed nearly all the malic acidpresent, with their fermentations showing a remainingG + F concentration of 144.9 and 129.9 g L)1 (Fig. 1),respectively. The corresponding acetic acid levels were,however, 0.70 and 0.64 g L)1. The latter results indicate

these strains would not be appropriate for use inconventional fermentations, although they might be ofservice in mixed or sequential fermentations. Table 1shows the final concentrations of lactic acid in thefermentations with the different yeasts; the absence ofmalolactic fermentation shows that no contaminationby lactic acid bacteria occurred.

Production of acetic acid

Differences in acetic acid production were seenbetween the yeast species, as well as between

40

90

140

190

240

0 5 10 15 20 25 30 35

Time (days)

G+F

(g L

–1)

7VA S6U 935 936 938 2139

b

a0

Figure 1 Consumption of glucose + fructose

by the studied yeast strains. Points are

means ± SD for three fermentations. Means

with the same letter are not significantly dif-

ferent (P > 0.05).

0.0

0.5

1.0

1.5

2.0

2.5

0 5 10 15 20 25 30 35

Time (days)

Mal

ic a

cid

(g L

–1)

7VA S6U 935 936 938 2139

a

d

c

b

Figure 2 Consumption of malic acid by the

studied yeast strains. Points are means ± SD

for three fermentations. Means with the same

letter are not significantly different

(P > 0.05).

New Schizosaccharomyces pombe applications S. Benito et al. 3

� 2012 The Authors International Journal of Food Science and Technology 2012


Schizosaccharomyces strains. Saccharomyces cerevisiae7VA and S. uvarum S6U produced mean acetic acidconcentrations of 0.23 and 0.36 g L)1, respectively(P < 0.05). The Schiz. pombe strains, however, pro-duced concentrations of 0.86–1.01 g L)1 (Fig. 3), ren-dering them unsuitable for use on their own inwinemaking. These results agree with those of otherauthors who report Schiz. pombe to be associated witholfactory defects (Unterholzner et al., 1988; Pitt &Hocking, 1999; Tristezza et al., 2010). The studiedSchizosaccharomyces strains are the only representa-tives of this species in the IFI collection, which incontrast has nearly 800 S. cerevisiae strains, highlight-ing the formers’ relative scarceness. All the presentSchizosaccharomyces strains were accidentally isolatedand certainly with no oenological criteria in mind. Anexhaustive study of the species might discover strainswith more moderate acetic acid production. Someauthors recommend biological malic deacidification beperformed with Schizosaccharomyces ssp. before theaddition of a selected Saccharomyces strain to avoidthis negative effect (Yang, 1973; Munyon & Nagel,1977).

Ammonia consumption

Ammonia is the primary nitrogen source for Saccharo-myces (Bell & Henschke, 2005). This would also appearto be true for Schizosaccharomyces as these strains allconsumed ammonia before PAN. A residual 4 mg L)1

of ammonia was recorded for all the studied yeasts,although their consumption kinetics were different(Fig. 4). Saccharomyces cerevisiae 7VA, S. uvarumS6U and Schiz. pombe 938 had consumed nearly allthe ammonia available by the 4-day check point, whileSchiz. pombe 2139, 936 and 935 did not consume it alluntil day 11.

Consumption of primary amino nitrogen

Saccharomyces cerevisiae 7VA and S. uvarum S6Ushowed minimum PAN concentrations of 23 and33 mg L)1, respectively, on day 4 (Fig. 5). However,the kinetics of the Schiz. pombe strains were slower;minimum concentrations of 54–60 mg L)1 were not seenuntil day 15. The PAN needs of this species thereforeseem to be smaller, probably due to its lower growthrate. An eventual increase in the PAN concentration wasseen in all fermentations, perhaps owing to autolysis atthe end of this process (Dizy & Polo, 1996; Fornairon-Bonnefond et al., 2002; Alexandre & Guilloux-Benatier,2006; Moreno-Arribas & Polo, 2009).

Pyruvic acid production

Saccharomyces cerevisiae 7 VA and S. uvarum S6Ushowed maximum pyruvic acid production at 4 days,reaching 0.061 and 0.045 g L)1, respectively (Fig. 6).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Time (days)

Ace

tic

acid

(g L

–1)

7VA S6U 935 936 938 2139

a

c

abb

d

ab

0 5 10 15 20 25 30 35

Figure 3 Acetic acid production by the stud-

ied yeast strains. Points are means ± SD for

three fermentations. Means with the same


(P > 0.05).

Table 1 Lactic acid content at the end of alcoholic fermentation.

Values are expressed as the means ± SD of three determinations

Yeast strain L-Lactic acid (g L)1)

Saccharomyces cerevisiae (7VA) 0.004 ± 0.002

S. uvarum (S6U) 0.006 ± 0.001

Schizosaccharomyces pombe (935) 0.002 ± 0.001

Schiz. pombe (936) 0.002 ± 0.001

Schiz. pombe (938) 0.001 ± 0.001

Schiz. pombe (2139) 0.001 ± 0.001




The Schizosaccharomyces strains produced more, how-ever, within the same timeframe and with significant(P < 0.05) differences between most of the memberstrains. Strain 938 reached a maximum of 0.386 g L)1

while 2139, 936 and 935 reached maxima of 0.292, 0.207and 0.199 g L)1, respectively. Pyruvic acid-based selec-tion studies on S. cerevisiae returned maximum valuesof 60–132 mg L)1 after 4 days of fermentation (Morata,2004) – values below those for the present Schiz. pombestrains. The same author earlier reported a strongcorrelation between the amount of pyruvic acid releasedinto the medium and the formation of vitisin A (Morata

et al., 2003). Strains with high pyruvate productionmight therefore be of interest in terms of pigmentproduction and stability.

Changes in colour intensity

A gradual decline in colour intensity was seen in allfermentations, but especially with S. uvarum S6U(Fig. 7). Final values of 8.26, 8.09, 7.78, 7.26 and 7.43OD were returned for Schiz. pombe 938, 936, 935,S. cerevisiae 7VA, S. uvarum S6U and Schiz. pombe2139, respectively (see graph for significant differences).

–10

10

30

50

70

90

110

Time (days)

Am

onia

(mg

L–1)

7VA S6U 935 936 938 2139

a

a

d

bc

b

0 5 10 15 20 25 30 35

Figure 4 Ammonia consumption by the

studied yeast strains. Points are means ± SD



(P > 0.05).

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

180.0

200.0

0 5 10 15 20 25 30 35

Time (days)

Prim

ary

amin

o ni

trog

en (m

g L–1

)

7VA S6U 935 936 938 2139

a

dc

abb

Figure 5 Primary amino nitrogen consump-

tion by the studied yeast strains. Points are

means ± SD for three fermentations. Means

with the same letter are not significantly dif-

ferent (P > 0.05).




Residual urea

The urea concentration (mg L)1) of the ‘wine’ wasdetermined at the end of fermentation. Saccharomycescerevisiae 7VA and S. uvarum S6U returned values of3.16 and 2.62 mg L)1, respectively (P < 0.05), while theSchizosaccharomyces strains all returned values of<0.5 mg L)1 (no significant difference between thesestrains, but P < 0.05 compared with the Saccharomycesspecies) (Table 2). Although none of these values arevery high, the lower values recorded for the Schiz. pom-be strains might be a consequence of their urease activity(Casas, 1999; Barnett et al., 2000; Deak, 2008). Ureaseactivity may be of interest with respect to wine safety asit removes urea, the precursor of ethyl carbamate

(Uthurry et al., 2004, 2006). These strains may also beof interest as they reduce the possibility of lactic acidbacteria growing by removing malic acid (another of

0.05

0.00

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0 5 10 15 20 25 30 35

Time (days)

Piru

vic

acid

(g L

–1)

7VA S6U 935 936 938 2139

a

dc

e

b

a

d

c

e

b

Figure 6 Production of pyruvic acid by the

studied yeas strains. Points are means ± SD



(P > 0.05).

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

Time (days)

ICM

7VA S6U 935 936 938 2139

abcbc

0 5 10 15 20 25 30 35

Figure 7 Change in must colour intensity over

fermentation with the studied yeast strains.

Points are means ± SD for three fermenta-

tions. Means with the same letter are not

significantly different (P > 0.05).

Table 2 Residual urea content after alcoholic fermentation. Values are

expressed as the means ± SD of three determinations. Means with the

same letter are not significantly different (P > 0.05)

Yeast strain Urea (mg L)1)

Saccharomyces cerevisiae (7VA) 3.16 ± 0.28a

S. uvarum (S6U) 2.62 ± 0.32a

Schizosaccharomyces pombe (935) 0.38 ± 0.18b

Schiz. pombe (936) 0.36 ± 0.24b

Schiz. pombe (938) 0.44 ± 0.31b

Schiz. pombe (2139) 0.32 ± 0.21b




their nutrient sources), thus reducing the risk of biogenicamine formation (Lonvaud-Funel, 2001; Alcaide-Hidal-go et al., 2007; De Fatima et al., 2007). Schizosacchar-omyces strains could therefore make fermentations saferfor human health by reducing the final urea content andavoiding malolactic fermentation.

Conclusions

The metabolic properties of Schiz. pombe, that is, thebreakdown of malic acid, production of pyruvic acid andthe breakdownof ethyl carbamate precursors, are of greatinterest in modern winemaking. However, its majordrawback is its strong acetic acid production at least forthe unselected strains commonly used in wine research.The selection of Schizosaccharomyces strains with low

production of acetic acid could bring a new oenologicaltool for unbalanced musts. This may help remove thespoilage stigma attached to this species. Other fermen-tation modalities such as mixed and sequential fermen-tation between Saccharomyces and Schizosaccharomycesto minimise the levels of acetic acid could be used.In addition, Non-Saccharomyces yeasts with high

pyruvic acid production, as the Schizossaccharomycesstrains studied in this research, can improve the forma-tion of stable pigments. The relevance of these com-pounds to increase chromatic parameters has largelybeen described in scientific bibliography.Finally, the selection of Schizosaccharomyces strains

with high urease activity can be developed as a new toolto assure wine safety.

Acknowledgments

This work was supported by the Ministerio de Ciencia eInnovacion (MCeI) (Project AGL2008-05603-C02-01 ⁄AGR). The authors are very grateful for the helpreceived from Biosystems S.A., and in particular toPablo Rodrıguez Plaza for the donation of the enzymekits used in this work.

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New applications for Schizosaccharomyces pombe in the alcoholic fermentation of red wines