10
Yeasts associated to Traditional Balsamic Vinegar: Ecological and technological features L. Solieri , P. Giudici Department of Agricultural Science, University of Modena and Reggio Emilia, Via J.F. Kennedy 17, 42100 Reggio Emilia, Italy Abstract Traditional Balsamic Vinegar (TBV) is an Italian homemade vinegar made with cooked grape must through a three-step process: conversion of sugars to ethanol by naturally occurring yeasts; oxidation of ethanol to acetic acid by acetic acid bacteria (AAB); and, finally, at least 12-years ageing. The cooked must is a selective and stressful medium for yeasts growth, due to its high sugar content and low pH values. Recent studies have shown that a large number of yeast species are involved in the fermentation, among them there are Zygosaccharomyces bailii, Zygosac- charomyces rouxii, Zygosaccharomyces pseudorouxii, Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces lentus, Hanseniaspora valbyensis, Hanseniaspora osmophila, Candida lactis-condensi, Candida stellata, Saccharomycodes ludwigii and Saccharo- myces cerevisiae. Nevertheless, the TBV-associated yeast population could be even more complex and many other slow-growing or poorly cultivable species might contribute to cooked must fermentation. In this review the main TBV yeast species are described, pointing out their role in TBV production and their influence on final product quality. Finally, both future developments in TBV yeast community studies (culture- independent and metagenomic techniques) and technological advances in TBV making (use of starter culture) are discussed. © 2007 Elsevier B.V. All rights reserved. Keywords: Vinegar; Yeast; Osmotolerance; Zygosaccharomyces; Hanseniaspora; Candida; Saccharomyces; Starter culture 1. Introduction Vinegar is a worldwide renowned acetic acid diluted solution, produced since ancient times with a double fermen- tation, alcoholic and acetic, from any fermentable sugary substrate. Balsamic vinegars, which are obtained by using cooked grape must, are just a small subset of the great variety of vinegars spread all over the world. There is a difference between Balsamic Vinegar and Traditional Balsamic Vinegar. The former is a category of industrial vinegars made by blending cooked must with wine vinegar and in some cases by adding a little amount of caramel; they are aged in barrels for a short time, generally from 2 months up to a few years. The latter, indicated here as TBV, is a category of high quality vinegars traditionally manufactured in Italian northern provinces of Modena and Reggio Emilia, following specific regulations, which are established by an Official Disciplinary (G. U. 124 of May 3, 2000). This product may be described as a highly dense and dark-brown vinegar aged at least for 12 years, having a sweet and sour taste, fruity and complex in flavour. Its origin arises from Middle-Age and it is largely spread still now and produced at family-owned small-scale. The chemical compo- sition of TBV is very heterogeneous because of its home-made preparation, but some common physico-chemical parameters are reported in Table 1. Recently TBV has been acknowledged by the European Union (CE n. 813/2000 April 17, 2000) as PDO product (protected denomination of origin). TBV is the product of a two-stage fermentation of cooked must: first a spontaneous alcoholic fermentation of sugars occurs, that is then followed by the acetic oxidation of ethanol. Even if acetic acid bacteria (AAB), that carry out acetic oxidation, have traditionally been considered to play the leading role in vinegar production, some studies have recently highlighted that also yeasts metabolism can affect TBV chemical properties in a remarkable way (Landi et al., 2005; Solieri et al., 2006). In fact yeasts are able to metabolise grape sugars and other compounds to ethanol, carbon dioxide and hundreds of secondary by-products that determine the Available online at www.sciencedirect.com International Journal of Food Microbiology 125 (2008) 36 45 www.elsevier.com/locate/ijfoodmicro Corresponding author. Tel.: +39 0522 522026; fax: +39 0522 522027. E-mail address: [email protected] (L. Solieri). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.06.022

Yeasts associated to Traditional Balsamic Vinegar: Ecological and technological features

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obiology 125 (2008) 36–45www.elsevier.com/locate/ijfoodmicro

International Journal of Food Micr

Yeasts associated to Traditional Balsamic Vinegar: Ecologicaland technological features

L. Solieri ⁎, P. Giudici

Department of Agricultural Science, University of Modena and Reggio Emilia, Via J.F. Kennedy 17, 42100 Reggio Emilia, Italy

Abstract

Traditional Balsamic Vinegar (TBV) is an Italian homemade vinegar made with cooked grape must through a three-step process: conversion ofsugars to ethanol by naturally occurring yeasts; oxidation of ethanol to acetic acid by acetic acid bacteria (AAB); and, finally, at least 12-yearsageing. The cooked must is a selective and stressful medium for yeasts growth, due to its high sugar content and low pH values. Recent studieshave shown that a large number of yeast species are involved in the fermentation, among them there are Zygosaccharomyces bailii, Zygosac-charomyces rouxii, Zygosaccharomyces pseudorouxii, Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces lentus,Hanseniaspora valbyensis, Hanseniaspora osmophila, Candida lactis-condensi, Candida stellata, Saccharomycodes ludwigii and Saccharo-myces cerevisiae. Nevertheless, the TBV-associated yeast population could be even more complex and many other slow-growing or poorlycultivable species might contribute to cooked must fermentation. In this review the main TBVyeast species are described, pointing out their role inTBV production and their influence on final product quality. Finally, both future developments in TBV yeast community studies (culture-independent and metagenomic techniques) and technological advances in TBV making (use of starter culture) are discussed.© 2007 Elsevier B.V. All rights reserved.

Keywords: Vinegar; Yeast; Osmotolerance; Zygosaccharomyces; Hanseniaspora; Candida; Saccharomyces; Starter culture

1. Introduction

Vinegar is a worldwide renowned acetic acid dilutedsolution, produced since ancient times with a double fermen-tation, alcoholic and acetic, from any fermentable sugarysubstrate. Balsamic vinegars, which are obtained by usingcooked grape must, are just a small subset of the great variety ofvinegars spread all over the world. There is a difference betweenBalsamic Vinegar and Traditional Balsamic Vinegar. Theformer is a category of industrial vinegars made by blendingcooked must with wine vinegar and in some cases by adding alittle amount of caramel; they are aged in barrels for a shorttime, generally from 2 months up to a few years. The latter,indicated here as TBV, is a category of high quality vinegarstraditionally manufactured in Italian northern provinces ofModena and Reggio Emilia, following specific regulations,which are established by an Official Disciplinary (G. U. 124 of

⁎ Corresponding author. Tel.: +39 0522 522026; fax: +39 0522 522027.E-mail address: [email protected] (L. Solieri).

0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2007.06.022

May 3, 2000). This product may be described as a highly denseand dark-brown vinegar aged at least for 12 years, having asweet and sour taste, fruity and complex in flavour. Its originarises from Middle-Age and it is largely spread still now andproduced at family-owned small-scale. The chemical compo-sition of TBV is very heterogeneous because of its home-madepreparation, but some common physico-chemical parametersare reported in Table 1. Recently TBV has been acknowledgedby the European Union (CE n. 813/2000 April 17, 2000) asPDO product (protected denomination of origin).

TBV is the product of a two-stage fermentation of cookedmust: first a spontaneous alcoholic fermentation of sugarsoccurs, that is then followed by the acetic oxidation of ethanol.Even if acetic acid bacteria (AAB), that carry out aceticoxidation, have traditionally been considered to play the leadingrole in vinegar production, some studies have recentlyhighlighted that also yeasts metabolism can affect TBVchemical properties in a remarkable way (Landi et al., 2005;Solieri et al., 2006). In fact yeasts are able to metabolise grapesugars and other compounds to ethanol, carbon dioxide andhundreds of secondary by-products that determine the

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Table 1Chemical characteristics of traditional balsamic vinegar (modified from Falcone et al., 2007)

Total solutes (Brix°) D-Glucose D-Fructose Glu+Fru Ratio Glu/Fru

Average 73.86 (1.73) 23.60 (3.45) 21.14 (3.37) 44.74 (6.25) 1.13 (0.21)

TA Ratio Brix /TA Acetate Malate Gluconate

Average 6.67 (0.88) 11.27 (1.53) 1.88 (0.45) 1.04 (0.32) 1.86 (1.28)

Amount expressed in g/100 g of Traditional Balsamic Vinegar, as average of values of 104 samples; Titrable acidity is expressed as g of acetic acid for 100 g of TBV.Abbreviations: Glu: glucose; Fru: fructose; TA: titrable acidity. In brackett standard deviation.

37L. Solieri, P. Giudici / International Journal of Food Microbiology 125 (2008) 36–45

properties of the fermented cooked must, that is used in thefollowing phases of acetic oxidation and ageing. However, inspite of the considerable interest of this topic, the influence ofyeasts on the fermented cooked must composition is still poorlydocumented and even controversial, especially with regard tothe effect of these microorganisms on the final TBV quality.

The understanding of TBV yeasts ecology and their geneticand physiological properties can provide new insights to controlthe fermentation process and the product features. As showed inother food-ecosystems, the conventional culture-dependentmethods, used so far to study TBV-associated yeasts, wouldaccess only a tiny subset of a large number of species. In the lasttwenty years, developments of culture-independent techniques,as well as the use of new genomically based approaches, haveimproved the inventory of food-related microbial species,clarifying their metabolic profiles (Tyson and Banfield, 2005).Reconstruction of individual and community metabolic net-works can elucidate the role of yeasts in TBV, as well as theirinteraction to each other and their environment. In addition,these techniques would help to determine the most suitableyeasts in TBV production, in order to select them as starterculture. The starter culture is the future challenge in TBV-making, as it would allow more efficient and predictable ethanolconversions, thus increasing the microbiological stability andprocess efficiency.

In this review we provided an overview of the relevantinformation concerning the main yeast species involved in TBVproduction and their advantages or disadvantages roles.Technological developments, such as selection of TBV-specificyeast starter culture, will also be considered, as well as futureperspectives for TBV yeast ecology studies.

2. Manufacturing process of TBV

TBV is obtained by a traditional slow process summarised inthree practical steps: cooking of must; microbiologicaltransformations (or two-stage fermentation) and ageing.

2.1. Cooking of grape must

Fresh must is obtained from a selection of local grapes, suchas Trebbiano, Lambrusco, Ancellotta, Sauvignon, Berzemino,Occhio di Gatta and Sgavetta, and it is brought to boiling pointin uncovered vessels. Impurities together with coagulatedproteins are removed by skimming, afterwards the temperatureis reduced and kept below 85–90 °C. The cooking is generally

stopped when the concentration of the must reaches 35–60° ofsoluble solids (°Brix). The formation of pigments by non-enzymatic browning and the degradation of hexoses bydehydration and cyclization, resulting in the formation of 5-hydroxymethyl-2-furaldehyde (HMF), are the main chemicalreactions which occur during the cooking of the must (Antonelliet al., 2004).

2.2. Microbiological transformations

After cooking, the medium is sterile and it is transferred intoopen wooden vessels, where an occasional contamination byyeasts occurs. The alcoholic fermentation is usually completedin a few weeks, during which yeasts increase rapidly, rangingbetween 102 to 106 colony-forming units/g (cfu/g) (Solieri et al.,2006). As alternative, the back-slopping practice is employed tospeed up the fermentation and avoid moulds growth. Neitherstarter culture nor dried yeast are used.

The second transformation is the bioconversion of ethanol toacetic acid by AAB, whose growth may occur spontaneouslyforming a thin layer or biofilm on the surface of fermentedcooked must, but it may be slowed down with ethanol values at9–10% or completely inhibited at higher ethanol values (Du Toitand Pretorius, 2002; Gullo et al., 2006). The oxidation can alsobe induced by manual transfer of the bacterial film from onevessel in active oxidation to a new steady one (Giudici et al.,2006). The result of this two stage fermentation is a vinegar richin sugar and ready for ageing.

2.3. Ageing

The final ageing is carried out in barrel sets of at least fivecasks of different sizes and woods (chestnut, oak and mulberrytrees are the most used), arranged in decreasing scalar volumes.Every year, the operation called “rincalzo” (refilling) isperformed: a small quantity of the aged vinegar is spilledfrom the smallest barrel, which contains the finished products.This barrel is then refilled with the content of the precedingbarrel and this operation is repeated, up to the first and largestcask, which receives new fermented and oxidised cooked must(Giudici and Rinaldi, 2007). The procedure allows to reintegratethe vinegar lost either by evaporation or by spilling. Beside thereactions that occur during the cooking of the grape must, otherimportant chemical changes take place during the ageing, suchas the formation of HMF and furan derivatives, owing to lowwater activity and pH (Muratore et al., 2006).

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38 L. Solieri, P. Giudici / International Journal of Food Microbiology 125 (2008) 36–45

3. TBV yeasts population

The alcoholic fermentation of grape juice is an ecologicallycomplex process studied in depth since Pasteur's times and,during the last 100 years, many reports have described thesuccession of various yeasts during wine fermentation,elucidating the role of non-Saccharomyces and Saccharo-myces yeasts (Amerine and Kunkee, 1968; Davenport, 1974;Kunkee and Amerine 1977; Fleet and Heard, 1993; Kunkee andBisson, 1993). However, despite of the growing economicimportance of TBV, the yeast population has been unknown fora long time. The first study dates back to the 1930s and it is apaper published by Sacchetti, later summarized in a book in1970 (Sacchetti, 1932, 1970), where the Author recognizedstrains belonging to genus Zygosaccharomyces (very similar tothose recognized as Z. rouxii, according to the latestnomenclature) as the predominant TBV yeasts and assumed acommensalistic interaction between Zygosaccharmyces yeastsand AAB. In the 1980s, Turtura and co-workers investigatedmore deeply the main TBV related species (Turtura, 1984, 1986;Turtura and Benfenati, 1988). They reported the presence of Z.bailii and Z. rouxii, identified on the basis of morpho-physiological features, such as the ability to grow at 1% aceticacid concentration. Afterwards, Giudici (1990) detected also theoccurrence of Saccharomycodes ludwigii strains, together withZ. rouxii and Z. bailii, and proposed the model of a two-stagefermentation, where first the yeasts convert sugar to ethanol andthen AAB grow and oxidize ethanol to acetic acid.

In the last 20 years, assignment of yeasts to species, generaand families has undergone a revolutionary change, thanks to theintroduction of PCR-based methodologies targeted to rDNAgenes and the use of sequence databases. Molecular methodshave also been successfully employed in yeast identificationfrom wine (Querol et al., 1992; Guillamon et al., 1998; Lopezet al., 2003), orange juice (Arias et al., 2002), cheese (Vasdinyeiand Deak, 2003; Lopandic et al., 2006), yoghurt (Caggia et al.,2001), cider (Morrissey et al., 2004; Valles et al., 2007) and frommany other fermented food and beverages (Beh et al., 2006). Themain phylogenetic markers employed are 26S (Kurtzman andRobnett, 1998), 18S (James et al., 1994) and 5.8S rRNA genes,together with two flanking internal transcribed sequences (ITS)(James et al., 1996). These molecular methods have providednew challenges to TBV-associated yeast studies and haveallowed both to increase the known species associated to cookedmust fermentation and to identify strains belonging to novelspecies. Recently Solieri et al. (2005, 2006) have employedmolecular techniques to characterize several strains isolatedfrom TBVs. They highlighted a complex yeast microflora,including not only Z. bailii, Z. rouxii and S. ludwigii, but alsoother Zygosaccharomyces species (Zygosaccharomyces mellis,Zygosaccharomyces pseudorouxii, Zygosaccharomyces bis-porus and Zygosaccharomyces lentus), two species belongingto Hanseniaspora genus (Hanseniaspora osmophila and Han-seniaspora valbyensis), two Candida species (Candida stellataand Candida lactis-condensi) and S. cerevisiae species.

All Zygosaccharomyces species associated to TBV arexerophilic yeasts growing in media with a high sugar

concentration (50 to 60%) and they are responsible for severalsugary beverages and food spoilage (Pitt, 1975; Fleet, 1992;Loureiro and Malfeito-Ferreira, 2003). Among TBVyeasts, it isremarkable that many strains belong to Z. lentus, a newosmotolerant species firstly described by Steels and co-workersin spoiled beverages, such as orange juice and in spoiled tomatoketchup (1998, 1999). TBV has been the isolation source of anew putative species, provisionally named Z. pseudorouxii,characterized by sequencing of D1/D2 26S rDNA and PCR-RFLP analysis of 5.8S-ITS region (Solieri et al., 2006). Thetaxonomic status of Z. pseudorouxii strains as new species orhybrid is still uncertain (James et al., 2005; Solieri et al., 2007).These strains showed a D1/D2 26S rDNA sequence differentfrom those of other Zygosaccharomyces species, together withat least two kinds of 5.8S-ITS regions. The presence of a uniqueD1/D2 26S rDNA gene, as well as intra-individual sequencepolymorphisms localized on ITS regions, induces to supposethat the mechanism of rDNA tandem copies evolution in Z.pseudorouxii may be non-concerted (Solieri et al., 2007).

Some other TBV-associated species, mainly Candida andHanseniaspora spp., are rarely detected in spoiled food andbeverages, but they are prevalently associated to early stages ofwine fermentation, and they can occur also during middle andlate phases. In spite of being a non-osmophilic species, S.cerevisiae has been found abundant and it has been detectedwith a high frequency in the lowest sugary cooked musts(Solieri et al., 2006), in accordance to Deak and Beuchat (1996)that found strains of S. cerevisiae able to grow in high sugarcontent food or beverages.

4. Methods of studying TBV yeasts: novel insights from“omics” approaches

Despite recent advances in understanding TBV microbialtransformations, knowledge of TBV-associated yeasts commu-nity is still partial. This lack greatly depends upon the use oftraditional culture-dependent approaches, including steps ofisolation, enumeration and identification of TBV yeasts. Thesephases show bias, which lead to a distorted view of the realyeasts ecosystem (Wintzingerode et al., 1997). For example,only predominant yeasts are recognized by using serialdilutions, whereas the less abundant types disappear. The agarplating technique is another major source of alterations, becauseit leads up to select only the easiest cultivable yeasts, while alarge majority of uncultivable and/or hardly cultivable micro-organisms are still unknown (Giraffa and Neviani, 2001).However slow-growing TBV yeasts, such as Zygosacchar-myces spp., Candida spp. and Hanseniaspora spp., that mightbe underestimated in general media, could be better recognizedby employing differential media and more favourable incuba-tion conditions (Beuchat et al., 2001; Loureiro and Malfeito-Ferreira, 2003). Other particular cautions, including the use of adiluent with a 10% sucrose concentration to avoid cell loss byosmotic stress, can be useful to obtain more realisticquantification of xerophilic TBV yeasts (Fleet, 1992).

The disadvantages briefly described above are intrinsic toyeast cultivation. Molecular culture-independent tools, such as

Page 4: Yeasts associated to Traditional Balsamic Vinegar: Ecological and technological features

Fig. 1. New strategy for yeast population study in Traditional Balsamic Vinegar: implementation of culture-dependent techniques (circumscribed in black line) withculture-independent and metagenomic analyses. A full-circle is obtained by community genomic data that increase both the cultivation strategies and sequencedatabase useful for probe-based analyses (modified from Tyson and Banfield, 2005).

Table 2Comparison among chemical properties of cooked must, grape juice, winevinegar and Traditional Balsamic Vinegar, affecting yeasts growth

Parameter Cooked must a Grape juice Wine vinegar b TBVc

Soluble solids(°Brix)

12.75–63.50 15–25 ∼7 69.0–75.5

pH 2.37–3.45 3.0–3.8 2.5–3.5 2.02–3.50Ethanol (%, v/v) 2.50–11.60 – b1.5 b1.5Titrable acidity(%, v/w)

0.72–5.08 0.65–0.90 6.0–7.4 N4.5–5.0

a Values referred to fermenting cooked musts, modified from Solieri et al.(2006).b Modified from Adams (1998).c Values referred to 50 not crystallized Traditional Balsamic Vinegar (TBV)

samples and modified from Falcone et al. (2007).

39L. Solieri, P. Giudici / International Journal of Food Microbiology 125 (2008) 36–45

DGGE, TGGE, in situ hybridization with oligonucleotideprobes, quantitative PCR, aim at characterizing the micro-organisms occurring in an environment without cultivation, andrely on the amplification and sequencing of single gene(ribosomal or conserved functional genes). These techniquesare successfully used in microbial studies of several food andbeverages (Ercolini, 2004; Beh et al., 2006), and may beemployed in cooked must ecological studies. A culture-independent technique, 16S PCR–DGGE, has been used tomonitor AAB population of TBV (De Vero et al., 2006), but hasnot been yet applied to TBV yeast ecology. Nevertheless, thesetechniques don't provide evidences about microbial metabolicfunctions, and thus cultivation-based studies are still afundamental step to understand yeast physiology and its impacton the chemical sensorial features of TBV.

A new experimental strategy to obtain a complete TBV-associated yeast community description may base on integra-tion among metagenomic tools, culture-dependent and culture-independent techniques. This approach is described in Fig. 1and it has been applied firstly to evaluate sea microbialcommunity (Tyson et al., 2004). In the case of TBV, as well inany food ecosystem, it could increase the yeast knowledge,improve information on metabolic profiles of hardly cultivableyeasts and optimise their cultivation conditions. The metage-nomic techniques are based on library construction andsequencing of DNA sequences obtained directly from TBVyeast consortium and they allow partial reconstruction of themetabolic network of whole yeast communities. This informa-tion may completely elucidate the influence of yeast metabolicactivities on cooked must properties and provide important

knowledge also about their nutritional requirements, so that thecultivation of poorly cultivable yeasts can be improved.

5. Factors affecting yeast survival and growth in grapecooked must

The yeasts living in grape juice are usually killed duringcooking within a few minutes at temperatures between 55 and65 °C. Therefore yeasts initially present in the cooked mustdepend on stochastic contamination events that occur aftercooking. Although, the intrinsic and extrinsic parameters ofcooked must determine a stressful environment for the survivalof the majority of the primary contaminant yeasts, so that onlyfew species can grow (Table 2). Sugar content, pH values

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40 L. Solieri, P. Giudici / International Journal of Food Microbiology 125 (2008) 36–45

(strictly related to weak acid content) and temperature aremainly critical factors for yeast growth.

5.1. Sugar concentration

Cooked must has a high sugar concentration (from 30 to 50%w/w) which decreases the aw to values lower than 0.9 (Table 2).The majority of yeasts requires a minimum water activity of0.85 and, at aw values between 0.61 and 0.75, only xerophilicfood spoilage yeasts can grow at slow rate. The yeasts mostfrequently isolated from high sugar content products belong tothe genus Zygosaccharomyces. Zygosaccharomyces bailii andZygosaccharomyces rouxii strains are the main spoiler in fruitjuices, sauces, carbonated soft drinks, salad dressings andketchup (Thomas and Davenport, 1985; Deak and Beuchat,1996). In particular Z. rouxii strains are able to grow at aw of0.62 in fructose solutions and of 0.65 in glucose/glycerolsolutions (Tilbury, 1980; Beuchat, 1983, 1987; Pitt andHocking, 1997).

The sugar hyper-osmotic stress responses have been well-documented in the non-osmophilic Saccharomyces cerevisiaespecies and involve the HOG pathways, general stress proteins(Msn2p/Msn4p-dependent genes) and the RAS-cAMP PKApathway (Hohmann, 2002; Erasmus et al., 2003). In osmoto-lerant yeasts regulatory circuits ruling osmotic response are stillpoorly known, even if it has been demonstrated that HOGresponse pathway and intracellular trehalose play a role in the Z.rouxii cell survival under high glucose concentration (D'Amoreet al., 1991; Iwaki et al., 1998; Smits and Brul, 2005).

5.2. pH and weak acids

The pH of cooked must is generally close to 3 (Table 2) anddoesn't inhibit the yeasts, which are acid tolerant and they cangrow at pH below 3. However weak lipophilic acids and low pHhave a synergic effect and reduce intracellular pH (pHi) belowthe normal physiological values, inhibiting the yeast growth.Moreover, acetic acid seems to directly affect transport orenzymatic activities, such as enolase, a key enzyme ofglycolysis (Pampulha and Loureiro-Dias, 1990).

The most pH-tolerant yeast is Z. bailii, followed by Z. rouxii.The latter is able to grow at a wide range of pH valuesdepending on sugar level: from pH 1.8 to 8.0 in highconcentrations of glucose or from pH 1.5 to 10.5 in a 12%glucose medium (Restaino et al., 1983; Tokuoka, 1993;Praphailong and Fleet, 1997; Membre et al., 1999). The newspecies, Zygosaccharomyces lentus, also detected in TBV, isable to grow at pH 2.2 (Steels et al., 1999). Another novelspecies, Zygosaccharomyces kombuchaensis, is resistant toacetic acid but sensitive to other weak acids (Steels et al., 2002).This finding has suggested that different resistance mechanismsexist in yeasts. Plasma membrane cation-translocating adeno-sinetriphosphatase (ATPase) is the most known mechanismthrough which yeasts maintain pHi homeostasis (Piper et al.,2001; Macpherson et al., 2005). However, Zygosaccharomycesspecies seem to have other more specific strategies, such asacetic acid–specific transporters, weak acid oxidative degrada-

tion and modification of membrane fatty acid composition(Golden et al., 1994; Sousa et al., 1996; for a review see Piperet al., 2001).

5.3. Temperature

Fermentation temperature is an important parameter affectingyeast growth rate and this response varies from species to species.Yeasts are mesophilic organisms with an optimum growingtemperature included between 20 and 40 °C. The role oftemperature in yeast ecology and in fermentation kinetics hasbeen widely studied in grape juice (Fleet and Heard, 1993), butpoorly in cooked must. When temperature decreases below 20 °Cin a wine fermentation, the contributions of non-Saccharomycescerevisiae yeasts (Hanseniaspora and Candida spp.) andcryotolerant Saccharomyces uvarum increase. At these tempera-tures, ethanol tolerance and growth rate of S. cerevisae are greatlyinhibited (Fleet, 1993; Rainieri et al., 1998).

Cooked must is stored at room-temperature, generally inAutumn and low temperatures can inhibit yeast growth anddetermine sluggish fermentations. Only the osmotolerantspecies Z. lentus has been described as suitable to grow atlow temperature (Steels et al., 1999). For other yeasts, theminimum temperature is higher in high sugary environments, inthe range from 12 to 13 °C for Z. bailii and from 6.5 to 10 °C forthe other species. However, during the full-fermentation phase,exothermic metabolic reactions increase the temperature in thevessels, that rises above 40 °C, thus reducing the yeast vitalityand ethanol yields. High temperatures above 40 °C inhibit themajority of yeast species growing in sugary and acidicenvironments, determining undesirable stuck fermentations.Strains of Z. bailii are reported to be able to grow at 35–37 °C(Barnett et al., 1990). Z. lentus can not grow at 30 °C (Steelset al., 1998, 1999).

6. Influence of yeast metabolism on TBV quality

As predominant microoganisms in cooked must, the yeastsactivity has three important consequences on TBV, including: i)fermentation of sugars to alcohols; ii) flavour production; andiii) chemico-physical transformations (Solieri et al., 2006). Fewstudies attempted to identify the yeast metabolic profiles incooked must. In addition, the TBV sensorial quality is the resultof a complex array of factors, generating not only from yeastsfermentation, but also from AAB oxidation and from chemicalreactions that occur during ageing. It is very difficult to separatethese contributions and evaluate their influences on finalproduct, even considering the long ageing time of at least12 years. The following summarizes the main developments inthis topic.

6.1. Ethanol production

Glycolitic production of ethanol from glucose and fructoseaffects the subsequent acetic oxidation in different ways. Lowethanol levels can inhibit acetic acid production and increaseincomplete oxidation of other polyols and sugars, mainly

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gluconic acid synthesis via direct glucose oxidation (Deppen-meier et al., 2002). However, ethanol has been also reported tobe responsible for yeast-AAB antagonism in TBV production(Giudici, 1990), because excessively high ethanol values (up to7–8% v/w) can inhibit AAB, specially at low pH values(Drysdale and Fleet, 1988). Finally, yeasts exose catabolismdecreases sugar values, affecting positively the followinggrowth of almost all AAB of TBV.

Some environmental parameters, such as oxygen and sugar,can affect the yeast ability to produce ethanol in cooked must. Insome cases oxygen has a positive role in wine fermentation,because it increases fatty acid and sterol biosynthesis andconsequently the yeasts viability (Alexandre and Charpentier,1998). The ability to carry out alcoholic fermentation despiteaerobic conditions (when sugar goes beyond a critical threshold,the so-called Crabtree effect) has been observed in some TBVspecies, such as Z. bailii (Merico et al., 2003) and in S.cerevisiae, but not in oxidative species, such as Candida spp.,which respire sugars in aerobic conditions. Therefore theoxygen level could negatively affect ethanol production whendominant species in cooked must are Crabtree-negative yeasts.

The ethanol production is influenced also by the sugarconcentration. Species with a weak fermentative power in grapemust, such as Z. bailii, Z. bisporus, Z. mellis, Z. pseudorouxii Z.rouxii and Candida spp., showed an ethanol production lessaffected by increases of sugar content in cooked must (Table 3).In particular, Solieri and Giudici (2005) found that Candidaspp. strains produced the highest ethanol concentration at thehighest sugar value tested. On the contrary, S. cerevisae strainsisolated from TBV showed a rapid decrease in ethanolproduction, if increasing the osmotic stress due to a highcooked must sugar content (Table 3).

6.2. Flavour compound production

Apart from ethanol, many secondary compounds areproduced during the alcoholic fermentation. The influence ofS. cerevisiae and non-Saccharomyces yeasts on the flavour ofwine and wine vinegar is well characterized (Ciani, 1998; Fleet,

Table 3Fermentation rate, ethanol production and selective sugar consumption of ten Traditi(from Solieri and Giudici, 2005)

Species Fermentation rate a Ethanol b

350 400 450 350

C. lactis-condensi 1.8 1.9 0.8 7.4C. stellata 1.8 1.7 0.6 7.3Z. bailii 0.6 0.7 0.4 7.0Z. pseudorouxii 0.3 0.2 0.3 6.3Z. mellis 0.4 0.4 0.3 5.7Z. bisporus 0.5 0.4 0.3 5.8Z. rouxii 0.4 0.4 0.4 5.3H. osmophila 1.4 1.5 0.7 5.0H. valbyensis 1.1 0.9 0.6 5.4S. cerevisiae 2.0 1.9 1.0 8.6a Fermentation rate evaluated as g of free CO2 for 100 ml cooked must after 72 hb Ethanol amount (v/v) evaluated at fermentation end at 350, 400 and 450 g/l sugc Residual sugars (g/l) evaluated at 350 g/l starting sugar. Abbreviations: Glu, glu

2003). C. stellata strains have been found to produce highglycerol, succinic acid, ethyl acetate and acetoin concentrationsthat positively influence the aromatic profile of wine vinegar(Ciani, 1998). Other two TBV-associated yeasts, H. osmophilaand S. ludwigii, produce high amount of ethyl acetate, acetoin,acetic acid and acetaldehyde and they are considered detrimen-tal yeasts in wine fermentation (Ciani and Maccarelli, 1998;Granchi et al., 2002). S. ludwigii has been proposed forcontinuous production of vinegar by Saeki (1990). The role ofyeast secondary metabolites in TBV sensorial quality has notyet been studied and a deeper knowledge in this topic isrequired.

6.3. Physical transformations

Yeast sugar catabolism exerts a strong influence even onsome physical properties of TBV (Landi et al., 2005; Solieriet al., 2006). Glucose crystallization is undesirable and it isnegative for the TBV quality. It can occur both in the barrels andin the bottles, causing heavy economical losses. Landi et al.(2005) have suggested a correlation among: i) the occurrence ofglucose crystal formation; ii) high sugar concentration ofcooked must; iii) the growth of fructophilic yeast species. Theincreased ratio between glucose and fructose in the cooked mustcould determine the glucose precipitation as solid crystals. Asshown in Table 3, many TBV yeast species preferentiallyferment fructose (fructophilic metabolism), leaving glucoseunfermented. C. lactis-condensi and C. stellata species havebeen found to have the highest fructophilic phenotype in cookedmust because they leave the initial glucose amount unchangedand they completely consume the fructose (Table 3). However,H. valbyensis, H. osmophila and S. cerevisiae showed aglucophilic phenotype both in wine (Ciani and Fatichenti,1999; Granchi et al., 2002) and in TBV (Landi et al., 2005;Solieri and Giudici, 2005). Similarly to Candida spp., also Z.bailii, Z. pseudorouxii, Z. mellis, Z. bisporus and Z. rouxiishowed a fructophilic metabolism (Table 3). The fructophilicbehaviour of Z. bailii is dependent on a fructose specific high-capacity system, while the glucose is transported by a lower-

onal Balsamic Vinegar — associated species at increasing sugar concentrations

Selective sugar consumption c

400 450 Glu Fru Glu/Fru ratio

8.2 7.3 171.7 1.4 122.107.8 6.8 151.8 9.7 15.657.2 5.9 138.6 15.0 9.246.5 6.3 159.9 29.8 5.366.0 5.7 175.6 35.8 4.906.1 4.6 155.8 51.1 3.055.7 5.3 160.6 53.2 3.025.3 4.4 98.5 133.9 0.755.2 4.0 101.2 127.3 0.798.2 5.9 42.3 96.4 0.44

at 350, 400 and 450 g/l sugars concentrations.ars concentrations.cose; Fru, fructose.

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Table 4Main fitness and quality traits useful for Traditional Balsamic Vinegar-specific yeast starter culture selection (modified from Solieri and Giudici, 2005)

Category Phenotypic traits Aims

Fitness Fermentation rate Quick fermentation; rapid implantation on the indigenous populationEthanol production Achievement of a sufficient amount of substrate for acetic oxidationOsmotolerance Growth in cooked mustCryotolerance/thermotolerance Growth in an temperature-uncontrolled environmentAcetic acid/low pH tolerance Growth in acidic environment

Quality Glucophilic metabolism No glucose crystallizationHigh fixed acidity production Increase of the acidity of TBVHigh acetic acid production Improvement of AAB growthInduction to AAB growth a Improvement of AAB growth

Abbreviation: AAB, acetic acid bacteria; TBV, Traditional Balsamic Vinegar.a Some yeasts have been found to increase the subsequent AAB growth and oxidation (Ciani, 1998). The mechanism of this stimulation is still unclear.

42 L. Solieri, P. Giudici / International Journal of Food Microbiology 125 (2008) 36–45

capacity system, partially inactivated by fructose (Pina et al.,2004). A similar mechanism could also occur in other Zygo-saccharomyces species.

7. From spontaneous fermentation to starter culture

Traditional practices for TBV production rely upon thegrowth of occasional contaminant yeasts in cooked must.However an uncontrolled fermentative process may frequentlyoccur, resulting in several disadvantages, such as stuck andsluggish fermentations; excessive ethanol production whichinhibits the subsequent acetic oxidation; increase of glucose/fructose balance with subsequent glucose crystallization. Theemploy of dry yeasts selected for the oenological field is uselessto overcome the drawbacks of the balsamic vinegar production,because S. cerevisiae wine strains are generally unable to growin high sugary cooked must. Some TBV producers haveoptimized spontaneous fermentation through back-slopping ofnew cooked must with small amounts of previously performedfermentation, to shorten the fermentation process and to reducethe risk of fermentation failure. This is an unconscious way ofusing a starter culture, because the best adapted species areimplanted over the indigenous population (De Vuyst, 2000).

Fig. 2. The main steps in a rational program of yeast starter culture selection for Tradi

Nevertheless, cooked must is still exposed to risks of alcoholicfermentation failure and following mould spoilage.

Technological developments in TBV making are directedwith increasing awareness to the use of yeast starter cultures,that can improve the safety, stability and efficiency of alcoholicfermentation. Giudici et al. (1992) have proposed the use ofselected starter cultures of Z. bailii to produce a high qualityfermented cooked must. Recent advantages in the yeastpopulation knowledge have highlighted that many otherdifferent yeasts can efficiently ferment the cooked must asdominant species (Solieri et al., 2006). In winemaking, theselection of starter culture is carried out within the species S.cerevisiae at strain level, whereas in TBV making more thanone species could be considered. Their metabolic profile incooked must is still unexplored and, consequently, their species/strain-specific influence on TBV quality is poorly determined.However it is possible to define the main features required toselect yeast species in TBV making. In Table 4 we summarizedthe TBV-specific phenotypical traits, divided into quality andfitness categories employable in a rational strain/speciesselection program (Solieri and Giudici, 2005).

The definition of required traits is only one of the many stepsin the complex process of TBV starter culture selection, as

tional Balsamic Vinegar fermentation (modified from Giudici and Solieri, 2006).

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43L. Solieri, P. Giudici / International Journal of Food Microbiology 125 (2008) 36–45

shown in Fig. 2. Considering the phenotypical behaviour of themajor TBV species, it is highly improbable that wild yeastspossess a casual recombination of all the desirable traits: severalspecies have some suitable traits, but none shows all desiredfeatures. An example is the occurrence of osmotolerance andglucophilic phenotype in TBVyeast species. The osmotoleranceis an essential trait to obtain a well-fermented cooked must, butthe most osmotolerant TBV-associated species show generally afructophilic metabolism, instead of a glucophilic feature, whichis necessary to reduce glucose crystallization. These findingssuggested that the selection of yeasts isolated from cooked mustis not a reliable practice to achieve a suitable starter culture forTBV, but it is only a starting point. Isolation of strains fromTBV allows to constitute a strains pull, on which genomicmanipulation technologies could be applied (Giudici et al.,2005). Moreover, osmotolerance and glucophilic metabolismare multigenic traits and knowledge of their genetics, by usingfunctional genomic techniques, could help to construct a yeastspecifically “for” TBV and not only “from” TBV.

Nowadays, starter cultures are not generally used in TBVproduction, because of the resistance of the operators tointroduce them in a rigorously traditional field. However, thedevelopment of starter cultures could improve the TBV quality,decrease the stuck fermentations and mould growth, as well asincrease successfully the growth of AAB, affecting positivelythe economical yield of TBV.

8. Conclusions

TBV is the product of different chemical, technological andmicrobiological contributions. Among the latter, yeasts have amain role in the first phase of alcoholic fermentation of thecooked must. Despite of recent advances in the knowledge ofTBVyeast microbiota, further investigations are necessary to: i)increase the number of species known to colonize the cookedmust; ii) improve the information about the yeast metabolicactivities affecting the TBV quality; iii) develop a new starterculture-based strategy in order to overcome unpredictablefermentation problems. For these aims, a polyphasic strategywhich integrates culture-independent, dependent techniques andmetagenomic approaches could be useful to understand the yeastcontribute to the production and to the final quality of TBV.However, at present, it is possible to have a preliminary and openlist of technological and quality traits for a starter selectionprogram, in order to construct yeasts specifically “for” TBV.

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