ru

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Received 10 May 2007; received in revised form 23 November 2007; accepted 30 December 2007

acterized by low fermentative capacity. After the first few daysof fermentation, they die off, due to the increasing concentration

Pina et al., 2004). Furthermore, several authors have reportedon the influence of non-Saccharomyces yeast species on winequality under usual winemaking conditions (Cabrera et al.,1988; Herraiz et al., 1990; Ciani and Picciotti, 1995; Lema

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International Journal of Food Microbiol1. Introduction

Spontaneous alcoholic fermentation of grape must is a com-plex process, carried out by sequential action of several yeastgenera and species, found on grapes and in must. Saccharo-myces cerevisiae is not the only species that grows duringnatural wine fermentations. The early stages in fermentation aredominated by the growth of non-Saccharomyces yeasts, char-

of ethanol (Heard and Fleet, 1985; Heard and Fleet, 1986).Subsequently, Saccharomyces, the most strongly fermenting,ethanol-tolerant species, take over the fermentation. Recentquantitative studies of winemaking ecology showed that sig-nificant levels of certain non-Saccharomyces species survive forlonger periods than previously thought (Fleet et al., 1984; Heardand Fleet, 1985; Cabrera et al., 1988; Pardo et al., 1989; Herraizet al., 1990; Lema et al., 1996; Egli et al., 1998; Fleet, 2003;Conventional wine yeasts produce high concentrations of volatile acidity, mainly acetic acid, during high-sugar fermentation. This alcoholicfermentation by-product is highly detrimental to wine quality and, in some cases, levels may even exceed legal limits. In this study, a non-conventional species, Torulaspora delbrueckii, was used, in pure cultures and mixed with Saccharomyces cerevisiae yeast, to ferment botrytizedmusts. Fermentation rate, biomass growth, and the formation of volatile acidity, acetaldehyde, and glycerol were considered. This studydemonstrated that T. delbrueckii, often described as a low acetic producer under standard conditions, retained this quality even in a high-sugarmedium. Unlike S. cerevisiae, this species did not respond to the hyper-osmotic medium by increasing acetic production as soon as it is inoculatedinto the must. Nevertheless, this yeast produced low ethanol and biomass yields, and the fermentation was sluggish. As a result, T. delbrueckiifermentations do not reach the required ethanol content (14%vol.), although this species can survive at this concentration. A mixed culture of T.delbrueckii and S. cerevisiae was the best combination for improving the analytical profile of sweet wine, particularly volatile acidity andacetaldehyde production. A mixed T. delbrueckii/S. cerevisiae culture at a 20:1 ratio produced 53% less in volatile acidity and 60% lessacetaldehyde than a pure culture of S. cerevisiae. Inoculating S. cerevisiae after 5 days' fermentation by T. delbrueckii had less effect on volatileacidity and acetaldehyde production and resulted in stuck fermentation. These results contribute to a better understanding of the behaviour of non-Saccharomyces and their potential application in wine industry. 2008 Elsevier B.V. All rights reserved.

Keywords: Wine; High-sugar fermentation; Torulaspora delbrueckii; Mixed culture; Volatile acidity; AcetaldehydeAbstractImpact of mixed Torulaspora delbculture on high-s

Marina Bely a,, Philippe Stoeckle a, Isabella UMR 1219 oenologie, Universit Bordeaux 2, INRA,

b ENITA de Bordeaux, 1 cours du Gnral de G Corresponding author. Tel.: +33 5 4000 6489; fax: +33 5 4000 6468.E-mail address: marina.bely@univ-pau.fr (M. Bely).

0168-1605/$ - see front matter 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2007.12.023ueckiiSaccharomyces cerevisiaegar fermentation

asneuf-Pomarde a,b, Denis Dubourdieu a

V, 351 cours de la Libration, 33400 Talence, Francele, CS 40201, 33175 Gradignan cedex, France

ogy 122 (2008) 312320www.elsevier.com/locate/ijfoodmicroet al., 1996; Ciani and Ferraro, 1998; Romano et al., 2003;Fleet, 2003). One of these non-Saccharomyces, Torulasporadelbrueckii (formerly Saccharomyces rosei), is reported to have

f Foa positive effect on the taste and aroma of alcoholic beverages(Herraiz et al., 1990; Moreno et al., 1991; Ciani and Picciotti,1995, Ciani and Maccarelli, 1998) and exhibits low produc-tion of acetaldehyde, acetoin, acetate, and ethyl acetate (Cabreraet al., 1988; Herraiz et al., 1990; Martinez et al., 1990, Ciani andFerraro, 1998). Due to its high fermentation purity, its use un-der standard conditions, in mixed or sequential culture withS. cerevisiae, has been proposed as a way of reducing the aceticacid content in wine (Herraiz et al., 1990; Ciani and Picciotti,1995; Ciani et al., 2006). Holm Hansen et al. (2001) indicatedthat T. delbrueckii was less tolerant to low available oxygenconditions than S. cerevisiae. On the other hand, little is knownabout the potential of non-Saccharomyces wine yeasts in high-sugar fermentations. Laffon-Lafourcade et al. (1981) evaluatedthe possibility of using S. rosei (T. delbrueckii) to improve thequality of botrytized wine. These wines are commonly madefrom grapes infected with Botrytis cinerea (noble rot), whichresults in concentrations of grape sugars ranging from 350to 450 g/L. Sipiczki et al. (2001), Naumov et al. (2002) andSipiczki (2003) reported a high frequency of Saccharomycesbayanus, which dominated the latter stages of fermentation, andthe presence of Candida stellata, a high acid producer in thefermenting yeast flora of Tokay wine. T. delbrueckii was alsodetected (1%) in botrytized must during the early stages offermentation (Sipiczki et al., 2001; Antunovics et al. 2003).

The amount of volatile acidity (mainly acetate) plays a sig-nificant role in wine aroma and excessive concentrations of thisalcoholic fermentation by-product are highly detrimental towine quality. The quantity of volatile acidity produced by S.cerevisiae is usually low (0.25 to 0.50 g/L expressed in aceticacid), but may be higher under certain fermentation conditions.In particular, during fermentation of high-sugar media, such asbotrytized musts, the volatile acidity content may be over 1.8 g/L or even higher, i.e. over the EEC legal limit of 1.5 g/L. S.cerevisiae produce acetic acid as a by-product of the hyper-osmotic stress response to the high-sugar concentrations ingrape must (Blomberg and Alder, 1992).

Recently Erasmus et al. (2004), comparing the suitabilityof different commercial S. cerevisiae wine yeast strains for ice-wine production, confirmed that wine yeast strains varied greatlyin their capacity to form acetic acid. The S. cerevisiae ZymafloreST strain was identified as one of the most suitable for ice-wineproduction, confirming previous findings in botrytized must(Barbe et al., 2000; Masneuf and Dubourdieu, 2000). It was alsorecently established that the initial assimilable nitrogen in themust and the physiological state of the inoculum affected thisstrain's volatile acidity production in high-sugar fermentations(Bely et al., 2003; Bely, Masneuf-Pomarde and Dubourdieu,2005). The optimum nitrogen concentration was 190mgN/L anddirect inoculation of rehydrated active dry yeasts produced themost volatile acidity, while yeast pre-culture for 24 h reduced thefinal production.

Nevertheless, in spite of more detailed knowledge of the S.cerevisiae Zymaflore ST strain, volatile acidity production in

M. Bely et al. / International Journal osweet wine may still be problematic, particularly in dry years inthe Sauternes area, when initial sugar concentrations are over350 g/L.The aim of this work was to evaluate the ability of a non-conventional, non-Saccharomyces yeast species, T. delbrueckii,often described as a low acid acetic producer, to ferment high-sugar must. This paper reports the impact of pure, mixed, andsequential cultures of T. delbrueckii and S. cerevisiae on fer-mentation behaviour, biomass growth, and analytical profile.

2. Materials and methods

2.1. Microorganisms and media

Two T. delbrueckii strains from the MUCL collection(Mycothque de l'Universit Catholique de Louvain-la-Neuve),T. delbrueckii 27828 and T. delbrueckii 31703, and one S.cerevisiae strain, Zymaflore ST (Laffort Oenologie, France), wereused. The Zymaflore ST strain was selected from native micro-flora during spontaneous fermentation of a botrytized must for itsability to produce small amounts of volatile acidity and SO2-binding compounds (Barbe et al., 2000, Masneuf and Dubour-dieu, 2000), and is commonly used in Sauternes.

Yeasts were grown at 30 C on complete YPD medium(1% yeast extract, 1% peptone, 2% dextrose) solidified with 2%agar.

2.2. Cell population count

Cell populations were determined by measuring absorbanceat 610 nm. One absorbance unit corresponded to 6106 cfu/mLfor the two T. delbrueckii strains and to 7106 cfu/mL for S.cerevisiae ST. Cell population was measured every day until nofurther increase in O.D was observed (O.D610 max.).

Yeast growth was also determined by plate counting in somecases. Samples were withdrawn throughout fermentation anddiluted appropriately in dilution medium. Non-Saccharomycescells were counted using lysine agar (LA) (Per litre: 66 g lysinemedium (Oxoid), 10 mL 50% potassium lactate, and 1 mL 10%lactic acid, pH 4.8). Total yeast cells were counted using YPDAmedium supplemented with chloramphenicol (100 mg/L). Thenumber of S. cerevisiae was given as the difference between thetotal plate count using YPDA and the plate count using LA.YPDA and LA plates were incubated at 25 C for 4 days beforecounting.

2.3. Must

The musts were obtained from botrytized Semillon grapes,harvested in vineyards in the Sauternes and Barsac appellations(20032005 vintages). They were collected in the cellar aftersettling and frozen. Initial sugar concentrations were 360 g/L.Musts were supplemented with nitrogen by adding ammoniumsulphate (NH4)2 SO4 to a level of 190 mgN/L. According toprevious studies (Bely et al., 2003), this nitrogen concentrationis optimum for minimising volatile acidity production by thisstrain. The indigenous yeast population was estimated by plate

313od Microbiology 122 (2008) 312320counting.Pasteurized musts were heated to 90 C for 15 min and the

effectiveness of this treatment was verified by plate counting.

of CO2 required (35 g/bottle, corresponding to an ethanol con-centration of approximately 14%vol.), while both T. delbrueckii27828 and T. delbrueckii 31703 stopped fermenting when only18.7 and 23.9 g/bottle CO2, respectively, had been released.The fermentation rate varied markedly depending on the yeastused (Fig. 1). Alcoholic fermentation with S. cerevisiae STwas completed 11 days after inoculation, whereas both T.delbrueckii 27828 and T. delbrueckii 31703 strains had slowerfermentation rates and fermentations stopped after 20 and26 days, respectively. It is worthwhile noting that, in both T.delbrueckii, the fermentation rate was the same during the first15 g/bottle CO2 released (corresponding to an ethanol contentof 6%vol.). S. cerevisiae ST had the highest cell densities (12.4)after 3 days (not shown), while both T. delbrueckii 27828 andT. delbrueckii 31703 stopped growing after 4 days' fermenta-tion at cell densities of 4 and 8.4, respectively (Table 1). Thisis consistent with previous results, which reported that T.

f Food Microbiology 122 (2008) 3123202.4. Fermentation conditions

Triplicate experiments were carried out in 375 mL sterilebottles (320 mL must per bottle) at 23 C, inoculated with 24 hpre-culture (grown in the same medium diluted 1:1 at 23 C)to obtain an initial level of 2106cells/mL. This pre-culturepreparation is essential to minimise volatile acidity productionin high-sugar fermentations (Bely et al., 2005). Mixedfermentation trials were simultaneously inoculated with 107

(or 5.106) T. delbrueckii 31703 cells/mL plus S. cerevisiaeST at ratios varying from 5:1 to 100:1. Sequential fermenta-tions were inoculated with 2106 cells/mL of T. delbrueckii,followed by 2106 cells/mL of S. cerevisiae after 5 days'fermentation.

Fermentations under static conditions were monitored byCO2 release, determined by measuring weight loss every 24 h.Fermentations were stopped by adding 250 mg/L SO2, when aminimumof 35 gCO2 per bottle had been released, correspondingto an alcohol concentration of approximately 14 %vol., in accor-dance with usual winemaking practices in Sauternes wineries.

2.5. Must and wine analysis

Ammonia was assayed by an enzymatic method (Roche-Biopharm), while primary amino acids were evaluated using anOPA/NAC spectrophotometric assay, as described by Dukesand Buzkle (1998).

Ethanol concentrations (%vol.) were measured by infraredrefractance (Infra Analyser 450, Technicon, Plaisir, France).Sugar (g/L) and volatile acidity (expressed in g/L acetic acid)were determined chemically by colorimetry (A460 nm) in con-tinuous flux (Sanimat, Montauban, France). Glycerol and acetal-dehyde were analysed by enzymatic methods (Roche-Biopharm,kit. n10148270035, 10668613035). These analyses werecarried out in the Sarco Laboratory (Floirac, France), accordingto quality assurance procedures approved by the French Ac-creditation committee COFRAC.

2.6. Statistical analysis

Volatile acidity, acetaldehyde, ethanol, glycerol, nitrogen, andsugar concentrations were subjected to single-factor varianceanalysis with three repetitions (=1%.), followed by a Newman-Keuls average comparison test (=5%) (ANOVA, StaboxSoftware , Grimmer Logiciels).

3. Results and discussion

3.1. Fermentation behaviour of pure culture

Several fermentations were carried out in a pasteurized mustcontaining 360 g/L sugar. The influence of different yeasts onfermentation kinetics and on the main oenological character-istics is shown in Fig. 1 and Table 1. All data correspond to the

314 M. Bely et al. / International Journal oaverage values obtained from triplicate fermentations. Ourresults on fermentation kinetics confirmed that S. cerevisiae STwas the only yeast capable of producing the minimum amountsdelbrueckii species had lower growth rates than S. cerevisiae(Mauricio et al., 1991; Ciani and Picciotti, 1995; Ciani, 1997).However, this reduced growth activity was not due to a deficitof assimilable nitrogen compounds, as they did not consume allthat was available. Similar behaviour was reported by Cianiet al. (2006).

The higher ethanol concentration in the S. cerevisiae STculture (15.1% vol.) was not the only difference. In comparisonwith the S. cerevisiae ST strain, the two T. delbrueckii fer-mentations also had a lower ethanol/sugar yield, which differsfrom a previous study using a low-sugarmust (Ciani and Picciotti,1995). Glycerol production by T. delbrueckii 27828 was lowerthan that of the other two yeasts.

T. delbrueckii 27828 and T. delbrueckii 31703 produced 2.7-and 1.9-fold less volatile acidity, respectively, than S. cerevisiaeST, confirming the specific characteristic of low acetic acidproduction of these yeasts species, as already reported in previ-ous studies under standard winemaking conditions (Cabreraet al., 1988; Herraiz et al., 1990; Martinez et al., 1990; Ciani andPicciotti, 1995; Ciani, 1997; Ciani and Ferraro, 1998).Fig. 1. Fermentation courses, in pasteurized must, at 23 C. Pure S. cerevisiaeST culture (S). Pure T. delbrueckii culture (T). Means of triplicate fermentations,max. SD0.9.

f FoYeast responds to increased external osmolarity by enhancedproduction and intracellular accumulation of glycerol to coun-terbalance the osmotic pressure (Blomberg and Alder, 1989;Blomberg and Alder, 1992; Myers et al., 1997). In S. cerevisiaeyeast, this regulation mechanism is due to the expression ofcertain osmoresponsive genes including glycerol 3-phosphatedehydrogenase, encoded by GPD1 (Albertyn et al., 1994;Attfied and Kletsas, 2000), as well as two types of aldhehydedehydrogenase, encoded by ALD2 and ALD3 (Navarro-Avinoet al., 1999; Erasmus et al., 2003; Pigeau and Inglis, 2005). Inorder to maintain the intracellular redox balance, yeast cellsregenerate an equimolar amount of cytoplasmic NADH. Thisrequirement seems to be partially met by a decreased reductionof acetaldehyde to ethanol, on the one hand, and an increasedoxidation to acetate, on the other (Blomberg and Alder, 1989).Thus, it is clear that overproduction of glycerol and acetate is

Table 1Main oenological characters of pure fermentations at 23 C

S. cerevisiae ST T. delbrueckii T. delbrueckii

27828 31703

Max.cell population(O.D610max)

12.4 4 8.4

Residual nitrogen (mgN/L) 12.001.41c 77.005.66b 100.502.12a

Residual sugar (g/L) 106.331.15c 219.633.79a 187.0010.82b

Ethanol (% vol.) 15.100.07a 7.400.21c 9.380.25b

Glycerol (g/L) 17.240.34a 14.050.92b 15.950.07a

Volatile acidity(g/L acetic acid)

1.170.08a 0.430.01c 0.620.01b

Acetaldehyde (mg/L) 80.716.31a 18.43.50b 27.003.00b

Yield ethanol/sugar (g/g) 0.48 0.43 0.46

Initial sugar concentration: 360 g/L. Initial nitrogen concentration: 190mgN/L.Initial glycerol concentration: 5.7 g/L.Means of triplicate fermentations SD. Values followed by different superscriptletters, within each line, are different according to theNewman-Keuls test (=5%).

M. Bely et al. / International Journal ounavoidable in high-sugar fermentation by S. cerevisiae (Caridiet al., 1999). The low volatile acidity production of T.delbrueckii and the non-negligible amount of glycerol suggestthat this species does not produce acetic acid as a by-product ofthe hyper-osmotic stress response to high-sugar concentrations.The direct consequence is a decrease in volatile acidity pro-duction. Actually, this species is known to be highly-resistant tostress conditions and is, therefore, often isolated in juice fruitsand bakery (Legan and Voysey, 1991), but there are no previousfindings concerning its resistance mechanism in high-sugarmedia.

In both T. delbrueckii trials, acetaldehyde production waslower than in pure S .cerevisiae fermentations, as reported byCiani and Picciotti (1995), Lema et al. (1996), Herraiz et al.(1990), and Ciani and Maccarelli (1998), but contrary to morerecent findings (Ciani et al., 2006). Acetaldehyde is an importantfermentation by-product, which affects wine quality. Barbe et al.(2000) stated that a high concentration of this compound in winefrom botrytized grapes reduced the amount of free SO2, an activeantimicrobial agent used to stop the fermentation. T. delbrueckii27828 and T. delbrueckii 31703 produced 4.4- and 3-fold lessacetaldehyde, respectively, than S. cerevisiae ST. It is worthpointing out that the S. cerevisiae ST strain was selected for itslow volatile acidity and acetaldehyde production (Barbe et al.,2000, Masneuf and Dubourdieu, 2000).

As expected, inoculating high-sugar musts with T. del-brueckii yeast resulted in stuck fermentations. Although thisyeast species had a low ethanol yield, it had the best secondarymetabolite profile. Low acetic acid and acetaldehyde productionis a positive feature for fermenting botrytized must, so a com-bination of T. delbrueckii species and S. cerevisiae was envis-aged to improve wine quality, while ensuring that fermentationwould be completed. T. delbrueckii 31703 was chosen for thefollowing study as it had a higher ethanol production.

3.2. Fermentation behaviour of mixed culture

Pure and mixed fermentations were carried out using T.delbrueckii 31703 and S. cerevisiae ST in pasteurized (PM) ornon-pasteurized must (NPM). Non-pasteurized musts were usedto mimic winemaking practices as closely as possible. Purecultures were inoculated with 2106 cells/mL. Mixed fermenta-tion trials were inoculated with 5.106 cells/mL of T. delbrueckii31703 and 106 cells/mL of S. cerevisiae ST (ratio T. delbrueckii/S. cerevisiae 5:1).

Yeast growth was monitored by plate counting. Non-Sac-charomyces cells (corresponding to T. delbrueckii 31703 inpasteurized must) were counted using lysine agar (LA). Thenumber of Saccharomyces cells was calculated as the differencebetween the total plate count using YPDA and LA. Inpasteurized must, the estimated Saccharomyces count corre-sponded to the S. cerevisiae ST population. The Saccharomycesand non-Saccharomyces populations in the non-pasteurizedmust were estimated at 2105 cfu/mL and 1.4104 cfu/mL,respectively.

The required amounts of ethanol (up to 14%vol.) were ob-tained in all fermentations except the pure T. delbrueckii 31703culture in PM, which stopped fermenting at 9.7%vol. (notshown). This result suggested that the indigenous yeastscontributed sufficiently to fermentation behaviour to producethe amount required of ethanol for the pure culture of T.delbrueckii 31703 in NPM. Fermentation time varied, depend-ing on the must treatment and yeast culture (Fig. 2). The fastestfermentation occurred in the pure S. cerevisiae ST culture inNPM, followed by S. cerevisiae ST in PM and mixed culture inNPM, then mixed culture in PM. The pure T. delbrueckii 31703cultures in NPM and PM took the longest to ferment (22 days).

As described above, the pure T. delbrueckii 31703 cultureshad the lowest maximum cell populations (O.D610 max.) andthe highest nitrogen residues (Table 2). However, nitrogenconsumption was higher in NPM, indicating competition be-tween T. delbrueckii 31703 and the indigenous yeasts in theNPM. On the contrary, in pure and mixed S. cerevisiae STcultures, irrespective of the must treatment, the nitrogen wasalmost completely consumed and maximum cell populationswere higher.

315od Microbiology 122 (2008) 312320Biomass evolutions during fermentation are shown in Fig. 3.No significant differences in the Saccharomyces populationswere observed between the pasteurized and non-pasteurized

These findings show that non-Saccharomyces species cansurvive at significant levels during high-sugar fermentation,

Fig. 3. Evolutions of the biomass in function of CO2 released at 23 C. Sac-charomyces populations (Fig. 3a) or non-Saccharomyces populations (Fig. 3b).Pasteurized (open symbols) or non-pasteurized must (filled symbols). Pure S.cerevisiae ST culture (S). Pure T. delbrueckii 31703 culture (T). Mixed T.delbrueckii 31703/S. cerevisiae ST (T/S). Means of duplicate fermentations,max. SD0.15.

316 M. Bely et al. / International Journal of Food Microbiology 122 (2008) 312320treatments (Fig. 3a). The maximum concentration of Sacchar-omyces cells varied from 6.9 to 7.9 Log (cfu/mL), with thehighest population in pure S. cerevisiae ST cultures (in NPM orPM) and the lowest in NPM inoculated with pure T. delbrueckii31703. Intermediate populationswere observed inmixed cultures,suggesting possible competition between non-Saccharomycesand Saccharomyces species, at critical nitrogen concentrations,which restricted Saccharomyces cell growth.

The biomass evolutions of non-Saccharomyces species(Fig. 3b) revealed similar maximum cell populations, 7.5 Log(cfu/mL), except for the pure S. cerevisiae ST inoculum. In PM,the pure T. delbrueckii 31703 culture slowly began to die offafter the level reached 6%vol. (corresponding to 12 g/bottleCO2 released), while in NPM the non-Saccharomyces yeastspopulation subsisted at a higher level, 6.6 Log(cfu/mL), untilfermentation was stopped. It is noteworthy that, in PM the pureT. delbrueckii 31703 culture stopped fermenting when 20 g/bottle CO2 had been released (at a cell concentration of 6.6 Log

Fig. 2. Fermentation courses at 23 C. Pasteurized (open symbols) or non-pasteurized must (filled symbols). Pure S. cerevisiae ST culture (S). Pure T.delbrueckii culture (T). Mixed T. delbrueckii 31703/S. cerevisiae ST (T/S).Means of triplicate fermentations, max. SD0.9.cfu/mL), while, in mixed culture, it was still present in similarcell concentrations at the end of fermentation. This suggeststhat, although T. delbrueckii 31703 is a low ethanol producer, itis able to survive at high ethanol concentrations, up to 14%vol.In NPM, the non-Saccharomyces cells in the mixed culture werepresent at maximum concentrations throughout fermentation.

Table 2Main oenological characters of pure and mixed fermentations at 23 C

Culture trial Max.cell populati

O.D610 max.

Pasteurized must Pure S. cerevisiae (S) 13.8Pure T. delbrueckii (T) 8.4Mixed S /T 14.2

Non-pasteurized must Pure S. cerevisiae (S) 14.8Pure T. delbrueckii (T) 9.6Mixed S/T 15.0

Initial sugar concentration: 360 g/L. Initial nitrogen concentration: 190mgN/L. InitiMeans of triplicate fermentations SD. Values followed by different superscript l(=5%).irrespective of the must treatment. In mixed culture in NPM, thenon-Saccharomyces and Saccharomyces cell populationsreached similar levels at the end of fermentation. The pure S.cerevisiae culture did not suppress the growth of indigenousnon-Saccharomyces yeasts. The reduced growth of Saccharo-myces species in mixed cultures, was the most significant

on Residual nitrogen Glycerol Volatile acidity

(mgN/L) (g/L) (g/L acetic acid)

16.670.58b 17.070.81a 1.000.06a

130.53.54a 14.700.53b 0.560.10b

11.001.00c 16.170.23a 0.730.09b

25. 330.58b 17.050.35a 0.940.06a

68.3310.02a 17.031.25a 0.790.02b

21.000.00b 16.400.99a 0.750.11b

al glycerol concentration: 6.6 g/L.Stuck fermentation.etters, within each column, are different according to the Newman-Keuls test

finding. These results differ from those reported by Nissen et al.(2004); Nissen and Arneborg (2003). In their research, theauthors found that, in mixed cultures, T. delbrueckii arrestedgrowth earlier than S. cerevisiae. This phenomenon was ap-parently due to a cellcell contact mechanism, dependent on thepresence of high concentrations of viable S. cerevisiae cells.These divergences were probably due to significant differ-ences in fermentation conditions: nitrogen, sugar, and inoculumconcentrations.

The main oenological characteristics of the fermentationsare presented in Table 2. The amount of glycerol was similar,except in the stuck fermentation with the pure T. delbrueckii31703 culture in PM.

There were no significant differences between the pasteur-ized and non-pasteurized treatments in terms of volatile acidityproduction, except for the T. delbrueckii 31703 trial in PM.

Saccharomyces and non-Saccharomyces populations of themust were estimated at 2104 cfu/mL and 2103 cfu/mL,respectively. Pure cultures were inoculated with 2106/mL.Mixed fermentation trials were simultaneously inoculated with107cells/mL T. delbrueckii 31703, together with S. cerevisiaeST in ratios ranging from 5:1 to 100:1. Sequential fermentationswere inoculated with 2106 cells/mL T. delbrueckii 31703 and2106 cells/mL S. cerevisiae ST after 5 days' fermentation,when 5%vol. of ethanol had been produced. CO2 release and themain oenological parameters are reported in Figs. 5 and 6 andTable 3.

The required amounts of ethanol (14%vol.) were obtained inall fermentations except the pure T. delbrueckii culture in PM andNPM, and the sequential fermentation (Table 3), which stopped at6.92, 10.47, and 13.15%vol., respectively. Fermentations using T.delbrueckii 31703, in pure or mixed cultures, had lower ethanol/sugar yields than those with S. cerevisiae ST alone. Fermentation

M. Bely et al. / International Journal of FoVolatile acidity concentrations in the wine varied considerably,ranging from 0.56 to 1 g/L acetic acid. Inoculation with a pureS. cerevisiae ST culture always produced more volatile acidity,irrespective of the must treatment. On the contrary, pure ormixed cultures with T. delbrueckii 31703 always produced lessvolatile acidity than pure S. cerevisiae ST. The volatile acidityproduction kinetics for one of the triplicate fermentations areshown in Fig. 4, revealing that the differences in final volatileproduction between the two pure cultures, mentioned above,were present from the onset of fermentation. In fact, in the pureS. cerevisiae ST culture, over 35% of the final amount wasproduced during the first few hours, when under 0.2 g/bottleCO2 had been released. In contrast, in the pure T. delbrueckii31703 culture, volatile acidity was produced gradually through-out fermentation. Intermediate behaviour was observed inmixed cultures, irrespectively of the must treatment. After aburst of volatile acidity production at the beginning of thefermentation, concentrations remained constant until 5 g/bottleCO2 had been released, then increased slowly until the end offermentation. Note that, after the first third of the fermentationperiod, the slopes of the volatile acidity/CO2 curves were simi-lar, regardless of the type of culture. All these results confirmed

Fig. 4. Volatile acidity productions in function of CO2 released, at 23 C.Pasteurized (open symbols) or non-pasteurized must (filled symbols). Pure S.

cerevisiae ST culture (S). Pure T. delbrueckii 31703 culture (T). Mixed T.delbrueckii 31703/S. cerevisiae ST (T/S). Means of duplicate fermentations.max. SD0.06.that T. delbrueckii 31703 does not response to increasedexternal osmolarity in the same way as S. cerevisiae ST, i.e. byproducing volatile acidity as soon as it is inoculated into a high-sugar must. The direct consequence is lower volatile acidityproduction than in pure S. cerevisiae ST cultures. In mixedcultures, the high T. delbrueckii 31703 concentration reducedacetate production during the first stage of fermentation.

The advantage of mixed culture was demonstrated in thisexperiment, as volatile acidity production was reduced, whilealcoholic fermentation was successfully completed in both NPMand PM. However, only one T. delbrueckii 31703/S. cerevisiaeST ratio was tested, so further experiments were carried out tooptimized this ratio.

3.3. Optimizing the mixed culture ratio

Pure cultures were carried out using pasteurized (PM) andnon-pasteurized must (NPM). In order to mimic winemakingpractice as closely as possible, mixed and sequential fermenta-tions were carried out in non-pasteurized must. The initial

Fig. 5. Fermentation courses at 23 C. Pasteurized (open symbols) or non-pasteurized must (filled symbols). Pure S. cerevisiae ST culture (S). Pure T.delbrueckii culture (T). Means of triplicate fermentations, max. SD0.6.

317od Microbiology 122 (2008) 312320rates varied depending on the must treatment (Fig. 5): fermenta-tions were slower in PM than NPM. This suggested that theindigenous yeasts contributed to fermentation kinetics, but did not

S. cerevisiae ST was unable to use the nitrogen available inthe must when it was introduced after 5 days' fermentation withT. delbrueckii 31703. The direct consequence was reducedgrowth cell, resulting in stuck fermentation. Further investiga-tion is required to clarify the mechanisms causing this reductionin S. cerevisiae ST fermentation capacity (presence of toxiccompounds, nutrient depletion, etc.).

Glycerol concentrations in wines fermented with pure S.cerevisiae ST and mixed cultures did not differ significantly. Onthe contrary, pure T. delbrueckii 31703 and the sequential fer-mentations produced less glycerol.

Once again, volatile acidity production by T. delbrueckii31703 in PM was very low compared to that of S. cerevisiae ST.In NPM, in both mixed and sequential cultures values werelower than those in pure S. cerevisiae culture, with a signifi-cant difference at 95% probability. Ciani et al. (2006) recently

Fig. 6. Fermentation courses, in non-pasteurized must, at 23 C. Pure S.cerevisiae ST culture (S). Pure T. delbrueckii 31703 culture (T). Mixed T.delbrueckii 31703/S. cerevisiae ST (T/S) with 107 T. delbrueckii 31703 cells/mL

318 M. Bely et al. / International Journal of Food Microbiology 122 (2008) 312320improve them sufficiently for the pure T. delbrueckii 31703culture to produce the required amount of ethanol, which differsfrom the previous result. This phenomenon was due to the lowerindigenous yeast concentration in this must (2.2104 cfu/mLcompared to 2.1105 cfu/mL).

In NPM, all mixed cultures had longer fermentation times,varying from 15 to 32 days, compared with 11 days for the pureS. cerevisiae culture (Fig. 6). The shortest multistarter fermen-tation was obtained with the highest concentrations of S.cerevisiae ST in the mixed inoculum.

As expected, regardless of the must treatment, the T.delbrueckii culture had the lowest maximum cell populationand the highest residual nitrogen. On the other hand, mixedcultures produced higher cell concentrations, reaching the samelevels as the pure S. cerevisiae ST culture at a T. delbrueckii31703/S. cerevisiae ST ratio of 5:1. The nitrogen residues

Means of triplicate fermentations, max. SD0.6.decreased in inverse proportion to the S. cerevisiae ST concen-tration in the inoculum. The pure T. delbrueckii 31703 andsequential culture exhibited similar behaviour, suggesting that

Table 3Main oenological characters of pure, mixed and sequential fermentations at 23 C

Culture trial Max.cellpopulation

Residualnitrogen

A610max (mgN/L)

Pasteurized must Pure S. cerevisiae 11.8 16.002.00Pure T. delbrueckii 6.2 107.0010.00

Non-pasteurized must Pure S. cerevisiae 11.2 21.672.08d

Pure T. delbrueckii 7.2 79.009.54a

Mixed T/S 5:1 11.1 23.671.53d

Mixed T/S 10:1 10.8 28.330.58cd

Mixed T/S 20:1 11.0 35.002.65c

Mixed T/S 50:1 9.4 45.003.61b

Mixed T/S 100:1 9.4 48.674.93b

Sequential T/S nd 85.005.66a

Initial sugar concentration: 360 g/L. Initial nitrogen concentration: 190mgN/L. InitiS. cerevisiae ST (S); T. delbrueckii 31703 (T); Mixed T. delbrueckii 31703/S. cerevMeans of triplicate fermentations SD. Values followed by different superscript l(=5%).reported similar results in pasteurized must containing lesssugar. There were similar reductions in mixed cultures at ratiosof 5:1 to 20:1, with up to 55% less volatile acidity in the wine. Insequential culture, volatile acidity production was only reducedby 37%. T. delbrueckii 27828 strain was also tested in sameconditions and comparable results were observed (not shown).

The significant variations were observed in acetaldehydeproduction, but these were not correlated with volatile aciditylevels. Concentrations varied from to 14.77 to 45.63 mg/L, withthe highest concentrations in both pure cultures of S. cerevisiaeST and T. delbrueckii 31703 in NPM. Acetaldehyde productionwas correlated with the proportion of S. cerevisiae ST in themixed cultures. Up to 68% less acetaldehyde was produced inmixed cultures with a T. delbrueckii 31703/S. cerevisiae STratio of 50:1. Recently, Cheraiti et al. (2005) showed up ac-etaldehyde exchanges between S. cerevisiae and S. uvarum inmixed culture under oenological conditions. Further investiga-tion is needed in our conditions.

The results show that, unlike pure T. delbrueckii, mixedcultures were capable of completing fermentation. Sequentialcultures gave the least positive results, in terms of volatileacidity and acetaldehyde production, while mixed cultures were

Ethanol Glycerol Volatile acidity Acetaldehyde Yieldethanol

(% vol.) (g/L) (g/L acetic acid) (mg/L) (g/g)

14.230.11 15.800.36 1.050.02 38.505.33 0.476.920.33 10.931.02 0.270.10 25.205.00 0.4414.100.35a 15.601.06a 0.890.16a 45.635.86a 0.4810.470.35c 13.470.38b 0.470.04cde 41.833.06a 0.4514.270.35a 16.000.10a 0.410.03e 30.904.00b 0.4614.430.04a 15.900.30a 0.400.03e 25.903.90b 0.4713.920.08a 15.070.91a 0.420.04de 18.201.47c 0.4714.150.10a 15.300.44a 0.500.01bcd 14.770.81c 0.4714.080.08a 15.901.51a 0.520.05bc 16.470.12c 0.4713.150.41b 13.170.15b 0.560.04b 20.301.84c 0.46

al glycerol concentration: 5.5 g/L.Stuck fermentation.

isiae ST (T/S) with 107 T. delbrueckii cells/mL.etters, within each column, are different according to the Newman-Keuls test

f Fothe most advantageous, irrespective of the S. cerevisiae con-centration. Final volatile acidity and acetaldehyde concentra-tions were up to 55% and 68% lower, respectively. Consideringthe reduction in both volatile acidity and acetaldehyde pro-duction, the best multistarter was T. delbrueckii 31703 mixedwith S. cerevisiae at a ratio of 20:1, with 107 T. delbrueckiicells/mL.

4. Conclusion

This study of fermentation in high-sugar must shows that T.delbrueckii is characterized by pure fermentation, with verylow volatile acidity and acetaldehyde production, confirmingprevious studies under standard winemaking conditions. Evenif it is a low ethanol producer, T. delbrueckii still has usefulpotential in sweet wine fermentation, as it does not seem torespond to osmotic stress in the same way as S. cerevisiae.Volatile acidity production remains constant throughout fer-mentation, in contrast to S. cerevisiae, where over 35% of thetotal production occurs in the initial stage of fermentation.Several studies have linked this overproduction by S. cerevisiaeto increased glycerol production, induced by osmotic stress.

One solution to the problem of excessive volatile acidityformation in high-sugar fermentations was to use mixed culturesof T. delbrueckii and S. cerevisiae, with a higher concentration ofT. delbrueckii to promote its growth. This mixed inoculum resultsin combined, rather than successive, fermentations, producinglower levels of acetic acid and acetaldehyde without affectingthe glycerol content. Reduction of the final volatile acidity andacetaldehyde productions up to 55% and 68% were obtained,respectively. This phenomenon depends on biochemical mechan-isms involving both yeast metabolisms. We show that a highpopulation of T. delbrueckii in mixed culture reduces the acetateproduction during the first stage of fermentation. Further inves-tigations are required to clarify the role of mixed culture inreducing volatile acidity production: could it be that the reducedgrowth of the S. cerevisiae induced by a high concentration ofT. delbrueckii, may explain the acetate reduction or are there anyacetate exchanges between the two yeasts?

Acknowledgements

The authors thank Chteau Rayne Vigneau for its collab-oration and acknowledge SARCO for technical assistance.

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Impact of mixed Torulaspora delbrueckiiSaccharomyces cerevisiae culture on high-sugar fermenta.....IntroductionMaterials and methodsMicroorganisms and mediaCell population countMustFermentation conditionsMust and wine analysisStatistical analysis

Results and discussionFermentation behaviour of pure cultureFermentation behaviour of mixed cultureOptimizing the mixed culture ratio

ConclusionAcknowledgementsReferences