MINI-REVIEW

Decontamination of ochratoxin A by yeasts: possible approachesand factors leading to toxin removal in wine

Leonardo Petruzzi & Milena Sinigaglia &

Maria Rosaria Corbo & Daniela Campaniello &

Barbara Speranza & Antonio Bevilacqua

Received: 5 March 2014 /Revised: 30 April 2014 /Accepted: 1 May 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Biological decontamination of mycotoxins usingmicroorganisms is one of the well-known strategies for themanagement of mycotoxins in foods and feeds. Yeasts are anefficient biosorbant, used in winemaking to reduce the con-centration of harmful substances from the must which affectalcoholic fermentation (medium-chain fatty acids) or whichaffect wine quality in a negative way (ethyl phenols andsulphur products). In recent years, several studies have dem-onstrated the ability of yeasts to remove ochratoxin A (OTA)by live cells, cell walls and cell wall extracts, yeast lees. Inspite of the physical and chemical methods applied to re-move the toxin, the biological removal is considered a prom-ising solution, since it is possible to attain the decontamina-tion without using harmful chemicals and without losses innutrient value or palatability of decontaminated food. Inaddition, adsorption is recognized as economically viable,technically feasible and socially acceptable. This paper in-tends to review the current achievements of OTA removalmediated by yeasts, the recent updates in the selection ofstrains acting at the same time as starters and as biologicaltools to remove OTA and the factors affecting the removalprocess.

Keywords Yeasts . OchratoxinA . Removal . Startercultures . Adsorbing tools

Introduction

The contamination of food and feed by mycotoxins [toxicmetabolites of fungi] has been recently characterized by theWorld Health Organization (WHO) as one of the most signif-icant sources of food-borne illnesses (Anly and Bayram2009); in fact, according to the Food and AgriculturalOrganization (FAO) of the United Nations, up to 25 % ofthe world’s agricultural commodities have been estimated tobe significantly contaminated with mycotoxins (Jard et al.2011). On the other hand, at least 100 countries have regula-tions regarding levels of mycotoxins in the food and feedindustry (van Egmond et al. 2007).

Currently, approximately 400 secondary metabolites withtoxigenic potential produced by more than 100 moulds havebeen reported (Jard et al. 2011), and this number is expected toincrease, probably due to the rising number of extreme weath-er events (Paterson and Lima 2011). Aspergillus, PenicilliumandFusarium fungal genera were considered the main sourcesof contamination (Reddy et al. 2010).

Some mycotoxins with a strong impact on human healthare aflatoxins (AFs), ochratoxin A (OTA), trichothecenes(deoxynivalenol (DON) and T-2 toxin), zearalenone (ZEN)and fumonisins (FMNs) (Afsah-Hejri et al. 2013).Unfortunately, these toxic compounds are generally thermo-stable and can remain present in crops even after all signs ofthe fungus itself have been removed (Inoue et al. 2013).

In recent years, OTA has received a special focus due to itstoxic effects and high incidence in a wide range of foods,including cereal products, coffee, spices, beer, grape and itsderivates and products of animal origin (Covarelli et al. 2012).

Ochratoxin A is a nephrotoxin with immunosuppressive,teratogenic and carcinogenic properties, as stated in 1993 bythe International Agency for Research on Cancer (IARC). Inhumans, OTA is frequently reported as the possible causativeagent of an endemic kidney disease observed in the Balkans

L. Petruzzi :M. Sinigaglia :M. R. Corbo :D. Campaniello :B. Speranza :A. Bevilacqua (*)Department of the Science of Agriculture, Food and Environment,University of Foggia, Via Napoli 25, 71122 Foggia, Italye-mail: antonio.bevilacqua@unifg.it

A. Bevilacquae-mail: abevi@libero.it

Appl Microbiol BiotechnolDOI 10.1007/s00253-014-5814-4

(Balkan endemic nephropathy and related urinary tract tu-mors) (Quintela et al. 2013).

Ochratoxin A is continuously recovered from the blood ofhealthy humans, and this datum confirms the continuousexposure to the toxin (Pozo-Bayón et al. 2012); a reason forthis high incidence is probably the importance of wine in thediet (ca. 13 % of the total dietary intake) (ScientificCooperation on Questions relating to Food-SCOOP, task3.2.7; European Commission 2002).

Some authors reviewed in the past the physical, chemicaland biological strategies to remove and/or decrease the con-tent of OTA (Shetty and Jespersen 2006; Amézqueta et al.2009; Abrunhosa et al. 2010; Quintela et al. 2013); however,there is no comprehensive report on yeasts as biological toolsto achieve the bioremediation of the toxin. Therefore, the aimof this paper was to specially focus on the role of yeasts asbiological and bioadsorbent tools to remove the toxin in wine,as well as on the factors acting on this complex phenomenon.

Basic facts on OTA contamination in grape product chain

The presence of OTA in grape juice and wine was reported forthe first time by Zimmerli and Dick (1995, 1996), and sincethen, the toxin was recovered in grapes, musts and wines inseveral Mediterranean countries such as France (Ospital et al.1998), Portugal (Ratola et al. 2004), Greece (Soufleros et al.2003), Italy (Brera et al. 2008) and Spain (Bellí et al. 2004).

Although Penicillium verrucosum and Aspergillusochraceus are considered to be the main OTA-producingspecies, there is strong evidence for the role of Aspergilluscarbonarius in OTA contamination in grape product chain(Quintela et al. 2013). A. carbonarius is a saprophyte in thetop layer of soil beneath vines and grows in berries injuredby biotic agents, pests and diseases, and by abiotic factors.The grape berry moth (Lobesia botrana) is a major cause ofcolonization by black Aspergillus species (section Nigri),due to its active role in transporting spores into injuredberries (Cubaiu 2008). The temperature, the moisture, theaeration, the period of infection and the interaction betweendifferent fungi play a significant role in mycotoxin diffusion(Esti et al. 2012).

The level of OTA relies upon the types of wines and vineproducts, regions and vintages (Solfrizzo et al. 2010). In fact,red wines have generally higher levels than white wines, dueto the increased time of contact between berry skins and grapejuice during the mashing stage (Cubaiu 2008). In addition, thecontent of the toxin is higher in sweet and special wines, ifcompared to dry ones due to the different oenological prac-tices (Covarelli et al. 2012).

A survey performed by the European Commission on1,470 samples revealed that the average value of toxin is0.36 μg l−1, although the wines from Southern Europe suffer

a higher contamination (15.6 μg l−1). High levels of OTAwerealso reported on dried vine fruits (e.g. sultanas and raisins), theoverall mean level being 3.10 μg kg−1 as a result of a surveyon 593 samples (European Commission 2002).

Based on the available scientific toxicological and expo-sure data, Regulation (EC) No. 123/2005 (EuropeanCommission 2005) set the limit of OTA to 2 μg l−1 for wine,grape juice, grape nectar and grape must intended for directhuman consumption; however, many trade agreements requirelower limits (e.g. 0.5 μg l−1) (Solfrizzo et al. 2010). On theother hand, the maximum level of OTA in dried vine fruitswas set to 10 μg kg−1 (European Commission 2005).

Measures for preventing OTA appearanceand possibilities of its removal

Both preventive and corrective approaches were proposed toreduce the incidence of OTA contamination; the preventionstrategies include the use of biocontrol agents and fungicidestowards A. carbonarius and insecticides against L. botrana.However, these approaches cannot completely prevent theproblem, and severe contamination of wine can occur espe-cially for some susceptible grape varieties in certain high-riskregions or vintages with climatic conditions promoting theinfection by A. carbonarius (Solfrizzo et al. 2010).

Physical decontamination of OTA involves first the remov-al of mouldy grapes or bunches before entering in thewinemaking process (Leong et al. 2006). According toQuintela et al. (2013), this method may be able to reduceOTA incidence up to 98 %, but it might not be economicallyfeasible for the wine industry.

Wine filtration through a 0.45-μmmembrane reduced OTAlevel by 80 %; on the other hand, a filtration through a 10-μmmembrane does not reduce significantly the level of the toxin(Quintela et al. 2013).

Another approach to remove OTA from contaminatedwine involves the use of inorganic adsorbent such as alumi-nosilicates, zeolites, bentonites, clays and activated carbon. Alimit for their use is that they could decrease the nutritivevalue and organoleptic properties and increase the cost offood production (Piotrowska et al. 2013). In addition, somefining agents commonly used in winemaking process couldcause adverse reactions in susceptible wine consumers(Quintela et al. 2013).

Oak wood fragments can be used to reduce the levels ofOTA in wine. The effectiveness of this treatment relies uponthe wood format (chips or powder), quantity, time of contactand wine composition (Quintela et al. 2013). The rules for theuse of this kind of approach were reported in CommissionRegulation (EC) No. 1507/2006 (European Commission2006); however, the use of oak wood fragments is forbiddenin some European regions (Quintela et al. 2013). Finally,

Appl Microbiol Biotechnol

chitosan, chitin, chitin glucan or chitin glucan hydrolysatefrom fungal origin may also be useful ancillaries for reducingOTA levels (Bornet and Teissedre 2008), but in some cases,they were responsible for a decrease of total polyphenols andtotal anthocyans (Quintela et al. 2013).

Bioremediation of OTA: scientific backgroundfor the application of yeasts as adsorbing tools

The ability to degrade OTA was observed for some bacteria(Streptococcus, Bifidobacterium and Bacillus) and fungi(Aspergillus, Alternaria, Botrytis and Penicillum genera). Inaddition, microbial-derived enzymes with carboxypeptidaseA activity (CPA) can also degrade OTA (Amézqueta et al.2009). However, there are still some concerns about the tox-icity of the products of enzymatic degradation and the unde-sired effects of non-native microorganisms on wine quality(Cubaiu 2008).

Another approach for the bioremediation is the adsorptionof OTA on cell surface/cell surface components (Amézquetaet al. 2009). Adsorption effects were reported forLactobacillus rhamnosus (Turbic et al. 2002), Lactobacillusspp. (i.e. L. acidophilus CH-5, L. rhamnosus GG, L.plantarum BS, L. brevis and L. sanfranciscensis)(Piotrowska and Zakowska 2005) and some wine lactic acidbacteria (LAB) (i.e. Oenococcus oeni RM11, L. plantarumCECT 748T and L. brevisRM273) (Del Prete et al. 2007). Thepeptidoglycan and polysaccharides are probably involved inthe toxin binding (Del Prete et al. 2007).

On the other hand, conidia of black Aspergillus spp. (i.e. A.niger, A. carbonarius and A. japonicus) showed adsorbingproperties toward OTA in grape juices and musts (Bejaouiet al. 2005).

Yeasts could retain different wine compounds; thus, theiruse as adsorbing tools to eliminate harmful and/or negativecompounds (medium-chain fatty acids, ethyl phenols andsulphur products) could be of interest (Jiménez-Moreno andAncín-Azpilicueta 2009).

Some researchers reported the idea that wine yeastscould efficiently remove OTA (Amézqueta et al. 2009);this idea was supported by five key factors: (1) it was notpossible to find wine products from the degradation ofOTA (Bejaoui et al. 2004; Cecchini et al. 2006), (2) acidor heat treatment retained the ability to bind the toxin(Bejaoui et al. 2004; Núñez et al. 2008), (3) the recoveryof OTA in yeast lees (Caridi et al. 2006; Cecchini et al.2006), (4) yeasts without cell wall (protoplast) were notable to adsorb the toxin (Piotrowska 2012) and (5) thefate of radiolabeled OTA during the fermentation of grapejuice (Lataste et al. 2004).

Generally, a surface phenomenon is the result of complexinteractions (van der Walls, resonance and electrostatic forces,

and hydrogen bonding) amongst the adsorbent, adsorbateand solvent. The molecular size and the physicochemicalproperties of OTA as well as the physical structure of theadsorbent, including the total charge and its distribution,the size of the pores and the surface area, play a signifi-cant role in OTA-binding by adsorbent materials (Huwiget al. 2001).

Why is there an interaction between OTA and the yeast cellwall? The answer relies upon the chemical traits of both ofthem. Ochratoxin A is a complex organic compound,consisting of chlorine-containing dehydroisocoumarin linkedthrough the 7-carboxyl group to 1-β-phenylalanine; phenoland carboxyl are the main functional groups involved in somedifferent adsorption mechanisms. First, OTA is consideredwith zearalenone as the less polar mycotoxin and could thenbe bound on hydrophobic surfaces through the phenol groupand via interactions of two-π-electron orbital. Moreover, thepKa of the carboxyl group of phenylalanine moiety is 4.4;thus, the toxin is partially dissociated at the pH of wine andcarries a positive charge on the amine function (NH3

+) (Leonget al. 2006).

Yeast biomass may be regarded as a good source ofadsorbent material, due to the presence in the cell wall ofsome specific macromolecules, such as mannoproteins andβ-D-glucans (Ringot et al. 2007). For example, the cellwall of Saccharomyces consists of two layers: an innerlayer made of β-1,3-glucan and chitin, which representsabout 50–60 % of the cell wall dry weight, and an outerlayer, which consists of β-1,6-glucan and heavily glyco-sylated mannoproteins. The mannoproteins are glycopro-teins having carbohydrate fractions made of around 98 %mannose and 2 % glucose; they are covalently linked to theinner cell wall layer, either directly to the β-1,3-glucanmatrix or indirectly via a β-1,6-glucan branch (Gonzalez-Ramos and Gonzalez 2006). This structure is highly dy-namic and strain-dependent, since about 1,200 genes drivethe synthesis of these cell wall components. Culture con-ditions, including pH, temperature, oxygenation rate, kindof medium and concentration or nature of the carbonsource, strongly modulate the quantity and structural prop-erties of β-D-glucans, mannans and chitin in cell walls;moreover, the cell cycle stage also interacts with the cellwall composition. For example, budding induces strongchanges in the distribution of the structural componentsof the cell wall such as chitin (Jouany et al. 2005).

Despite the strong evidence of OTA adsorption to cellwalls (Bejaoui et al. 2004; Garcia-Moruno et al. 2005;Leong et al. 2006; Ringot et al. 2007; Núñez et al. 2008;Bizaj et al. 2009; Piotrowska 2012), Angioni et al. (2007)found that some yeasts could reduce OTA levels in wine,although they did not adsorb OTA; therefore, they suggestedthe existence of a possible pathway for the degradation of thetoxin, different from the mechanism involving L-β-

Appl Microbiol Biotechnol

phenylalanine and ochratoxin α; however, they could not findany product of hydrolysis.

Application of yeasts or yeast-derived productsas adsorbing tools to remove OTA from syntheticand natural grape juices, wines or model wine systems

Some studies focused on the reduction of OTA in syn-thetic and natural grape juices, wines or model winesystems by yeasts or inactive dry yeast (IDY) preparations(i.e. inactive yeast, yeast autolysates, yeast extracts andyeast hulls or walls) (Pozo-Bayón et al. 2009); Table 1reports a short overview of the available references. Theexperiments were carried out under laboratory conditions,and viability was not a prerequisite to achieve the removalof the toxin. Indeed, yeasts were intended simply asadsorbing tools.

The first comprehensive report on the use of yeasts andyeast-derived products intended as winemaking tools for theremoval of OTAwas conducted by Bejaoui et al. (2004). Theyreported that heat- and acid-treated cells were able to bindsignificantly higher levels of OTA than the viable ones in asynthetic grape juice medium. Viable yeasts bound up to 35%of OTA, depending on yeast concentration, whilst heat- andacid-treated cells decreased the toxin by 90.80 and 73 %,respectively. In addition, a comparative experiment betweenheat-treated cells and commercial yeast walls, at two differentconcentrations (0.2 and 6.7 g l−1) in a contaminated grapejuice, showed the highest efficiency by heat-treated cells ableto completely remove the toxin in 5 min. This trend could bedue to the fact that heating might cause changes in the surfaceproperties of cells, like the denaturation of proteins or theformation of Maillard reaction products. On the other hand,acidic conditions could affect polysaccharides by releas-ing monomers, which are further fragmented into alde-hydes after the breaking of glycosidic bonds. Thesereleased products could harbour higher adsorption sitesthan viable cells with an enhancement of OTA removal(Piotrowska et al. 2013).

Several winemaking practices involve a prolonged contactbetween yeasts or IDY preparations and wine (i.e. ageing onlees and the production of sparkling wines), thus suggestingthat yeast cells could play a significant role in OTA removalalso at the end of the fermentation process (Núñez et al. 2006).During this period, some genes encoding proteins involved incell wall biosynthesis are strongly up-regulated (fks1, gsc2,ssd1 and mpt5) (Pradelles et al. 2008); in addition, variableamounts of mannoproteins and glucans can be released intowines due to yeast autolysis interacting with wine phenoliccompounds, decreasing their astringency and/or acting asprotective colloids, and enhancing the color stability of redwines (Del Barrio-Galán et al. 2012). Mannoproteins can also

interact with OTA, as recently demonstrated by Núñez et al.(2008).

The most recent winemaking technologies are focusedon the use of vinification on lees (used successfully withwhite wines) for red wines (Garcia-Moruno et al. 2005);Garcia-Moruno et al. (2005) reported that OTA removalwas different using white and red lees wine. After 7 days ofcontact (20 g l−1 of lees), the white lees wine reduced OTAby 70.90 % whilst the red lees wine decreased the toxin by51.50 %. After 80 days. OTA reduction was ca. 73.90 %with the white lees and 63.10 % with the red ones. Thisdifference was attributed to competition on the bindingsites between OTA and polyphenols, and this hypothesiswas supported by Meca et al. (2010), who reported thatphenols, proteins, organic acid and colloidal particlesshowed the same ability of OTA to react with differentcompounds of the cell wall.

Another trend is the use of commercial yeast derivativepreparations, as an alternative to lees; these products couldreduce the time required to obtain wines with physico-chemical and sensory characteristics similar to those agedon lees (Del Barrio-Galán et al. 2012). Piotrowska et al.(2013) added a thermally inactivated biomass ofSaccharomyces cerevisiae yeast to wines from white grapeand blackcurrant juices and decreased the content of OTAby ca. 60 %. The heat treatment (85 °C for 10 min) wasalso proposed by Núñez et al. (2008) to increase the re-moval yield of whole yeast cells and yeast cell walls in amodel system up to 95 %. According to Piotrowska et al.(2013), the use of inactivated yeasts as OTA-adsorbingtools might be highly advantageous since such biomassdoes not change organoleptic features of end products; onthe other hand, Núñez et al. (2008) reported that heat- andacid-treated cells could negatively affect the quality ofwine. Nevertheless, the existence of potential unexpectedeffects related to odorant compounds initially present in theIDY preparations (Pozo-Bayón et al. 2009) should be care-fully evaluated.

Since the natural autolysis is a long-lasting process, it isusual to use model systems to obtain results in shorterperiods of time (Martínez-Rodríguez et al. 2001); Petruzziet al. (2014a) tested two commercial (BM45 and RC212)and three wild S. cerevisiae strains (W13, W47 and Y28)and a commercial cell wall preparation to remove OTA. AspH, temperature and ethanol are the major factors affectingautolysis in a model system, different pHs (3.0 and 3.5)temperatures (25 and 30 °C) and ethanol concentrations (5,10 and 15 %) were used. They reported that yeasts removedthe toxin by 3.69–81.87 %; S. cerevisiae Y28 showed thehigher removing percentages (24.60–81.87 %), whereas thestrain RC212 removed up to 25 % of the toxin. On the otherhand, yeast cell walls reduced OTA by 2.47–50.00 %. Thedecrease of the toxin was higher at 30 °C and pH 3.0 and

Appl Microbiol Biotechnol

with 15 % of ethanol. The viable count of yeasts was belowthe detection limit after 3 days in all the samples, thussuggesting that the bioremediation was not strictly relatedto cell viability.

Two wild strains (S. cerevisiae W47 and Y28) werepreviously studied for their ability to remove OTA in alaboratory medium (Petruzzi et al. 2013); the strainsdecreased the toxin by 36–42 % in a medium adjusted

Table 1 Review of wine yeasts and yeast-derived products used as adsorbing tools to remove ochratoxin A (OTA) from synthetic and natural grapejuices, wines or model wine systems

Yeast or yeast-derived product

State of cells Type of assay Experimental conditions Adsorbentamount(g l−1)

OTA level(μg l−1)

OTA removal(%)

References

Yeast cell walls Not treated Red wine Incubation at 25 °C 0.5–1.0 1.9 12–30 Silva et al. (2003)

Saccharomycescerevisiae

Viable Synthetic grape juice Incubation for 2 h, 30 °C 6.7–27.4 2 17.00–35.00 Bejaoui et al. (2004)

Heat-treated Synthetic grape juice Incubation for 2 h, 30 °C 6.7–27.4 2 75.00–90.80

Acid-treated Synthetic grape juice Incubation for 2 h, 30 °C 6.7 2 73

Heat-treated Red juice Incubation for 2 h, 30 °C 0.2–6.7 10 100

Yeast wallsadditive

Not treated Red juice Incubation for 2 h, 30 °C 0.2–6.7 10 100

Saccharomycescerevisiae

IY Red wine Stirring for the first 90 minand then stored for7 days at ca. 20 °Cwithout further shaking

1–4 3.04–3.30 25–60.30 Garcia-Moruno et al.(2005)

Saccharomycescerevisiae

Lees from whitemust fermentation

Red wine Stirring for the first 90 minand then stored for 7 or80 days at ca. 20 °Cwithout further shaking

20 1.65–7.09 70.90–73.90

Lees from red mustfermentation

Red wine Stirring for the first 90 minand then stored for 7 or80 days at ca. 20 °Cwithout further shaking

20 2.49–4.12 51.50–63.10

Yeast hulls Not treated Red wine Incubation for 2 days, atca. 22 °C

2.00–5.00 5 28.00–43.00 Leong et al. (2006)

NI IY Red wine Stirring for 90 min andthen stored for 30 or90 min at ca. 20 °Cwithout further shaking

1–4 2.55–2.78 20.00–46.50 Savino et al. (2006)

IY Red wine Stirring for the first 90 minand then stored for 7 or80 days at ca. 20 °Cwithout further shaking

4 2.84 62.00–81.00

Saccharomycescerevisiae

Rehydrated IY Model wine Incubation for 214 h 1 10 0.8 Núñez et al. (2008)

Rehydrated IYandheat-treated

Model wine Incubation for 214 h 1 10 95.4

Yeast cell walls Not treated Model wine Incubation for 214 h 1 10 18.8

Heat-treated Model wine Incubation for 214 h 1 10 95.0

Candida spp.,Kloeckera spp.,Rhodotorulaglutinis,Cryptococcuslaurentii

Biomass fromculture broth

White wine Incubation for 4 h, 25 °C NI 10 4.75–21.40 Var et al. (2009)

Biomass thermallyinactivated

White wine Incubation for 4 h, 25 °C NI 10 8.08–30.45

Saccharomycescerevisiae

Biomass fromculture broth

Model wine Incubation for 9 days at 25or 30 °C, with ethanolcontent of 5, 10 or15 %, at pH 3.0 or 3.5

10 2 3.69–81.87 Petruzzi et al. (2014a)

Yeast cell walls Not treated Model wine Incubation for 9 days at 25or 30 °C, with ethanolcontent of 5, 10 or15 %, at pH 3.0 or 3.5

10 2 2.47–50.00

Saccharomycescerevisiae

Biomass thermallyinactivated

White wine Incubation for 24 h, 30 °C 0.005 1,000 64.40 Piotrowska et al. (2013)Red wine Incubation for 24 h, 30 °C 0.005 1,000 62.40

IY inactive yeast, NI not indicated

Appl Microbiol Biotechnol

to pH 3.5, containing 10 % of ethanol and incubated at37 °C.

Based on available data, we could suppose that somevariables play a major role in OTA removal efficiency of yeastderivatives, i.e.:

& The enzymatic and chemical protocol for their production,as the enzymatic and thermal procedure currentlyemployed for their production could have a strong influ-ence on their physical and compositional characteristics(Núñez et al. 2006);

& The amount (Silva et al. 2003; Bejaoui et al. 2004), al-though most of the researchers performed the experimentsusing amounts of yeast cell wall preparations of up to0.40 g/l, i.e. the legal limit set by Commission Regulation(EC) No. 1410/2003 (European Commission 2003);

& The physiological state of yeast cells before the produc-tion of autolysates, as the content of mannoproteins andglucans is usually higher in the lysates from viable cellsthan from rehydrated cells (Guilloux-Benatier andChassagne 2003).

Application of yeasts to remove OTA throughout alcoholicfermentation and factors affecting its removal

Some basic requirements for an ideal method to remove OTAare the low cost, the simplicity of use and the absence ofeffects on the quality of wine (Amézqueta et al. 2009).Unfortunately, inactivated cells or IDY products could nega-tively affect the quality of wine (Núñez et al. 2008; Pozo-Bayón et al. 2009), whilst the use of viable cells does not showthis drawback; moreover, active yeasts have a long history ofsafe use as starter cultures (Abrunhosa et al. 2010).

Over the last years, some authors identified yeasts fromSaccharomyces and non-Saccharomyces genera able to de-crease OTA content by 0.60–100 % throughout fermentation(Table 2). Some key factors able to influence this phenomenonare the conditions for the assay (laboratory or industrial scale,in vitro or in vivo conditions), kind of the strain, cell dimen-sion, flocculence, cell sedimentation kinetics and toxin con-centration (Caridi et al. 2006; Caridi 2007; Cecchini et al.2006; Angioni et al. 2007; Ponsone et al. 2009; Petruzzi et al.2014b, d).

Kind of fermentation, scale and OTA content

The level of OTA in wine is significantly lower than in grapes,due to toxin removal throughout vinification, and especiallyduring alcoholic fermentation, as a result of yeast activity and/or the interaction between OTA and grape constituents (Paster2008). In red vinification, most of OTA is removed in the

solid–liquid separation stages, when the wine or the juice isseparated from the skins (Bizaj et al. 2009). In white vinifica-tion, grapes are pressed before fermentation and OTA mightbind more effectively to the grape proteins and solids, whichare removed during clarification. In addition, other steps (e.g.additional clarification steps) allow higher OTA-binding rates(Paster 2008). We could suppose also an interaction betweenOTA and the natural microbiota of grape must, probablycontributing to the modification of the removal yield of inoc-ulated starter strains.

Differences in OTA removal may be related to the scale ofassay, as the management of the fermentation could be diffi-cult in macro-scale experiments; on the other hand, changes inOTA content during small-scale experiments might be simplymanaged.

Under laboratory conditions (from 10 ml to 2 l), OTAwasremoved by 32.6–90.95 %, depending on the strain and themedium (Caridi et al. 2006; Meca et al. 2010; Cecchini et al.2006; Esti et al. 2012).

OTA removal was also found in higher volumes. Fernandeset al. (2007) reported that the toxin was reduced by 55.5–77.7 % in microvinification trials consisting of 20 kg ofgrapes; however, the mass balance performed in eachvinification trial revealed that this decrease was duepredominantly by the adsorption onto suspended solids inmusts and wines. Ponsone et al. (2009) showed that a signif-icant reduction of OTA (i.e. 77–100 %) after fermentation in100-l tanks was caused by an extensive adsorption onto thesolid parts of the grapes and yeast lees. Only one study wascarried out at industrial scale (800-hl tanks) by Grazioli et al.(2006); they reported that maceration increased OTA contentby 45 %, whilst alcoholic fermentation caused its reduction(30 %).

Concerning the effect of the initial amount of OTA,Piotrowska et al. (2013) studied OTA removal by two strainsof S. cerevisiae: Malaga LOCK 0173, able to remove OTA(1,000μg l−1) by 82.80, 10.70 and 35.40%, respectively, fromwhite juice, blackcurrant juice and synthetic medium, and thestrain Syrena LOCK 0201 respectively reducing toxin con-centration by 85.10, 65.20 and 21.00 %. Csutorás et al. (2013)reported that a commercial S. cerevisiae type “Fermol PremierCru” removed OTA (initial content 4,000 μg l−1) by 90, 85and 73 % from red, rose and white must, respectively.

Chemico-physical factors acting on OTA removal

Although many papers focused on the role of some physico-chemical factors on the course of wine fermentation (Belyet al. 2003, 2008; Hernández-Orte et al. 2006; Torija et al.2003), few data are available on their influence on the removalof OTA by yeasts. This issue is of great concern consideringthat some oenological parameters can modify the surfaceproperties of cell wall (Vasserot et al. 1997); thus, they could

Appl Microbiol Biotechnol

Tab

le2

Reviewof

wineyeastsableto

removeochratoxin

A(O

TA)throughout

alcoholic

ferm

entatio

n

Yeasts

Num

berof

strains

Type

ofassay

Scaleof

assay

Ferm

entativeconditions

OTA

level

(μgl−1 )

OTA

removal

(%)

Oenologicaltraitsof

yeasts

References

Saccharomyces

uvarum

,Saccharomyces

cerevisiae,

Saccharomyces

bayanus,Kloeckera

apiculata,Torulaspora

delbrueckii,

Schizosaccharomyces

pombe,C

andida

pulcherima,

Saccharomycodes

ludw

igii

1Whiteandredmusts

Laboratoryscale

NI

240.10–72.12

/Morassutetal.(2004)

NI

1Red

mustfrom

TintaRoriz,

TourigaNacional,

TourigaFranca,Tinta

Barroca

andTintoCão

Microvinification

T=25

°C4.0

90/

Ratolaetal.(2005)

Saccharomyces

sensu

stricto

20Whitemustn

aturally

contam

inated

with

OTA

Laboratoryscale

T=25

°C1.58

39.81–90.95

Ethanol

productio

nCaridietal.(2006)

Whitemustadded

with

OTA

Laboratoryscale

T=25

°C7.63

67.89–83.34

Ethanol

productio

n

Saccharomyces

cerevisiae,

Saccharomyces

cerevisiae×

Saccharomyces

bayanus,

Saccharomyces

bayanus,Kloeckera

apiculata,Torulaspora

delbrueckii,

Schizosaccharomyces

pombe,C

andida

pulcherima,

Saccharomycodes

ludw

igii

1WhitemustfromTrebbiano

ToscanoandMalvasia

delL

azio

Laboratoryscale

T=20

°C,S

=22.28°Brix

246.83–52.16

/Cecchinietal.(2006)

Red

mustfrom

Primitivo

Laboratoryscale

T=20

°C,S

=20.79°Brix

253.21–70.13

/

Saccharomyces

cerevisiae

1Red

mustfrom

Negroam

aroand

Primitivo

Industrial-scale

vinificatio

nT=20–32°C

0.88

30/

Graziolietal.(2006)

Saccharomyces

cerevisiae

4Sy

nthetic

medium

Laboratoryscale

T=25

°C,S

=200gl−

10.25–18.52

28.00–48.00

Ethanol

productio

nOrroetal.(2006)

Kloechera

apiculata

1Sy

nthetic

medium

Laboratoryscale

T=25

°C,S

=200gl−1

0.25–18.52

32–49

Ethanol

productio

n

Saccharomyces

cerevisiae

7Sy

nthetic

medium

Laboratoryscale

T=25

°C,S

=200gl−1

0.20–6.00

28–100

Ethanol

productio

nAngioni

etal.(2007)

Kloechera

apiculata

7Sy

nthetic

medium

Laboratoryscale

T=25

°C,S

=200gl−1

0.20–6.00

25–64

Ethanol

productio

n

Saccharomyces

cerevisiae

1Red

mustfrom

Vinhão

Microvinification

T=18

±1°C

0.43–7.48

55.50–77.70

/Fernandesetal.(2007)

Saccharomyces

cerevisiae

1Red

must

Laboratoryscale

T=25

°C0.41

41Ethanol

productio

nLasram

etal.(2008)

Rosemust

Laboratoryscale

T=25

°C0.41

44Ethanol

productio

n

Appl Microbiol Biotechnol

Tab

le2

(contin

ued)

Yeasts

Num

berof

strains

Type

ofassay

Scaleof

assay

Ferm

entativeconditions

OTA

level

(μgl−1 )

OTA

removal

(%)

Oenologicaltraitsof

yeasts

References

Saccharomyces

cerevisiae

1Sy

nthetic

medium

Laboratoryscale

T=20

°C,S

=180gl−1

4.95

2.60

Sugarconsum

ptionand

volatileacidity

productio

n

Bizajetal.(2009)

Saccharomyces

cerevisiae

1Sy

nthetic

medium

Laboratoryscale

T=20

°C,S

=180gl−1

3.48

2.00

Sugarconsum

ptionand

volatileacidity

productio

nSaccharomyces

cerevisiae

3Whitemustfrom

Moscato

ofSaracena

Laboratoryscale

T=16–18°C

,S=38°B

rix

10>40

/Blaiotta

etal.(2009)

Saccharomyces

cerevisiae

2Red

mustfrom

Tempranilo

Microvinification

T=24–28°C

,S=

24.70°Brix

683–86

Sugarconsum

ption,

ethanoland

acids

productio

n

Ponsoneetal.(2009)

Red

mustfrom

Bonarda

Microvinification

T=24–28°C

,S=

21.50°Brix

0.3

77–100

Sugarconsum

ption,

ethanoland

acids

productio

nSaccharomyces

cerevisiae

16Whitemustfrom

Moscato

Laboratoryscale

T=25

°C,S

=22°B

rix

1032.60–50.40

/Mecaetal.(2010)

Saccharomyces

cerevisiae

48Whitemustfrom

Zibibbo

Laboratoryscale

T=25

°C4.10

7.80-81.95

/Caridietal.(2012)

Saccharomyces

cerevisiae

1Whitemustfrom

Sauvignonblanc

Laboratoryscale

T=20

°C,S

=20.5°B

rix

1562

/Estietal.(2012)

Saccharomyces

cerevisiae

1 1Red

mustfromCesanesedi

Affile

Laboratoryscale

T=20

°C,S

=22°B

rix

1579.00

/

Whitemustfrom

Sauvignonblanc

Laboratoryscale

T=20

°C,S

=20.5°B

rix

1567.00

/

Saccharomyces

cerevisiae

Rosemust

Microvinification

T=12

°C10–4,000

83.00–86.00

/Csutorásetal.(2013)

Whitemust

Microvinification

T=12

°C10–4,000

73.00–76.00

/

Red

must

Microvinification

T=12

°C10–4,000

88.00–90.00

/

Saccharomyces

cerevisiae

1Sy

nthetic

medium

Laboratoryscale

T=30

°C,S

=20

gl−1

1,000

35.40

/Piotrowskaetal.(2013)

Whitejuice

Laboratoryscale

T=30

°C,S

=12°B

rix

1,000

82.80

/

Blackcurrantjuice

Laboratoryscale

T=30

°C,S

=12°B

rix

1,000

10.70

/

Saccharomyces

cerevisiae

1Sy

nthetic

medium

Laboratoryscale

T=30

°C¸S=20

gl−1

1,000

21.00

/

Whitejuice

Laboratoryscale

T=30

°C,S

=12°B

rix

1,000

85.10

Ethanol,glyceroland

acidsproductio

nBlackcurrantjuice

Laboratoryscale

T=30

°C,S

=12°B

rix

1,000

65.20

Ethanol,glyceroland

acidsproductio

nSaccharomyces

cerevisiae

2Sy

nthetic

medium

Laboratoryscale

T=25

or30

°C,S

=200or

250gl−1

25.41–49.58

Sugarconsum

ption,

ethanol,glycerol

and

volatileacidity

productio

n

Bevilacqua

etal.(2014)

Saccharomyces

cerevisiae

1Sy

nthetic

medium

Laboratoryscale

T=25

or30

°C¸S=200or

250gl−1,N

=250or

310mgl−1

26.00–25.66

Sugarconsum

ption,

ethanoland

glycerol

productio

n;phenotypicaltraits

Petruzzietal.(2014b)

Saccharomyces

cerevisiae

2Sy

nthetic

medium

Laboratoryscale

T=25

or30

°C,S

=200or

250gl−1,N

=250or

310mgl−1

22.82–27.53

Sugarconsum

ption,

ethanoland

glycerol

Petruzzietal.(2014c)

Appl Microbiol Biotechnol

affect the binding capacity of yeasts. Petruzzi et al. (2014b)studied the kinetics of OTA removal by a potential starterstrain (i.e. S. cerevisiae W13) as affected by the most impor-tant environmental parameter (e.g. temperature) (Pizarro et al.2008), as well as by two major nutrients acting on the fate ofmust fermentation (sugar and nitrogen) (Heinisch and Rodicio2009). At this scope, laboratory-scale fermentations werecarried out at two temperatures (25 and 30 °C) and sugarlevels (200 and 250 g l−1), with or without supplementationof medium with diammonium phosphate (DAP) (300 mg l−1).The yeast was able to reduce OTA up to 57.21 %, with apositive effect of temperature and sugar. These resultsconfirmed the data by Patharajan et al. (2011), who studiedsix different temperatures (10, 15, 20, 25, 30 and 35 °C) tooptimize OTA removal in a liquid medium by three yeasts(Metschnikowia pulcherrima MACH1, Pichia guilliermondiiM8, Rhodococcus erythropolis AR14) and found the highestremoval at 30 °C. Petruzzi et al. (2014b) suggested that themajor ability to remove OTA at 30 °C could be associated tothe release of cell wall polysaccharides. The effects of sugarconcentration could be related to the level of mannoproteins,as these compounds are strictly dependent on sugar (Mecaet al. 2010). Finally, Petruzzi et al. (2014b) found that theremoval was highest after 3 days of fermentation; then, thetoxin was partially released in the medium and the perfor-mances of the strain decreased (i.e. 6.0–25.66 % reduction). Arelease of toxins after their removal was also recovered forother compounds; Peltonen et al. (2001) reported the releaseof aflatoxin B1 (AFB1) by lactobacilli (i.e. L. amylovorus andL. rhamnosus). They suggested that this phenomenon couldbe associated to the weak bond in lactobacilli/AFB1 com-plexes. Since hydrogen bonding and ionic or hydrophobicinteraction seem to be related to OTA adsorption mechanismby S. cerevisiae (Cecchini et al. 2006), Petruzzi et al. (2014b)considered this statement as a possible explanation for OTArelease by yeasts.

In another study, Petruzzi et al. (2014c) tested two wildS. cerevisiae strains (W28 and W46) under the same experi-mental conditions reported for the strain W13. As a result, thestrains W28 and W46 were able to decrease the toxin by ca.70 %, with the highest removing effect observed after 3 daysat 30 °C in the presence of 250 g l−1 of sugars and with DAP;after 10 days, the toxin was partially released into the mediumand the performances of the strains decreased (i.e. 2.82–27.53 % reduction). As reported above, sugar and temperatureplayed a major role, with a significant effect of DAP supple-mentation, too. As regards nitrogen, we could suppose that thestrains exhibited a significant ability to remove OTA onlywhen the conditions were closer to the optimal ones (i.e.greater YAN content in the must), probably due to a higherrelease of cell wall polysaccharides (Giovani et al. 2010). Thedifferent trends of the three strains (i.e. W13, W28 and W46)toward DAP addition could be due to the strong strainT

able2

(contin

ued)

Yeasts

Num

berof

strains

Type

ofassay

Scaleof

assay

Ferm

entativeconditions

OTA

level

(μgl−1 )

OTA

removal

(%)

Oenologicaltraitsof

yeasts

References

productio

n;phenotypicaltraits

Saccharomyces

cerevisiae

4Sy

nthetic

medium

Laboratoryscale

T=25

or30

°C,S

=200or

250gl−1

20.60–42.80

Sugarconsum

ption,

ethanol,glycerol

and

volatileacidity

productio

n;phenotypicaltraits

Petruzzietal.(2014d)

NInotindicated,/

none

oenologicaltraitassessed¸Ttemperature,S

sugarcontent,Nnitrogen

contentasyeastassim

ilablenitrogen

(YAN)

Phenotypicaltraitsincludethefollo

wing:resistance

tosingleandcombinedstressconditions,enzymaticactiv

ities,hydrogensulphide

productio

n,interactionwith

phenoliccompounds

andbiogenicam

ines

form

ation;

1°B

rixmeans

1weightp

ercentageof

reducing

sugars

Appl Microbiol Biotechnol

dependence on the response to changes in environmentalconditions (Giovani et al. 2010).

OTA removal as a new trait to select functional yeast startercultures

Recently, a new approach for the study of OTA-removingyeasts was proposed by Petruzzi et al. (2014b, c, d), i.e. yeaststrains intended as functional starter cultures, as they showboth the benefits of traditional starters and a health- orproduct-focused function. In this sense, a drawback in theliterature is that few data are available on the oenological traitsof OTA-removing yeasts (see Table 2), whilst this aspect is ofgreat concern for the possible selection of functional strainsacting at the same time as starters and as biological tools toremove the toxin.

Thus, the removal of OTA could be an interesting andattractive trait to complement the classical oenological char-acterization based on technological and qualitative key traits,such as the tolerance and high ethanol production, exhaus-tion of sugars, growth at high sugar concentration, goodglycerol production, growth at high temperatures, low hy-drogen sulphide and volatile acidity production, resistance tosulphur dioxide and good enzymatic profile (Nikolaou et al.2006). This idea is supported by the fact that the removal oftoxin by yeasts is genetically controlled and is a polygenicinheritable trait of wine yeasts (Caridi et al. 2012). However,the toxin could be released back into the medium after itsadsorption (Bevilacqua et al. 2014; Petruzzi et al. 2013,2014b, c), probably due to the nature of the adsorptionmechanism involved (hydrogen bonding, ionic or hydropho-bic interaction) (Cecchini et al. 2006). Strain dependencecould play a significant role also on the stability of thecomplex cell/toxin, as well as on the yeast cell wall compo-sition in response to a variety of fermentative/growth condi-tions (Bevilacqua et al. 2014).

Therefore, Petruzzi et al. (2014d) used a polyphasic ap-proach, consisting of the genotypic identification and theevaluation of the phenotypic traits and fermentative perfor-mances in a model system (temperature, 25 and 30 °C; sugarlevel, 200 and 250 g l−1), to select wine starters of S. cerevisiaefrom 30 autochtonous isolates from Uva di Troia cv., a redwine grape variety of the Apulian region (Southern Italy). Theability to remove OTAwas used by the authors as a desirablefunctional trait to improve the safety of wine. As a result, 11biotypes were identified and a strain, representative of eachbiotype, was further studied. Four strains (Y20, W21, W40and W41) were able to reduce OTA by 0.60–42.80 %; inaddition, the strains W21 and W40 were promising in termsof ethanol, glycerol and volatile acidity production, as well asfor their enzymatic and stress resistance characteristics. In thissense, interesting traits were also possessed by S. cerevisiaestrains W13 (Petruzzi et al. 2014b), W28 and W46 (Petruzzi

et al. 2014c), e.g. high tolerance to single and combined stressconditions; β-D-glucosidase, pectolytic and xylanase activi-ties; low-to-medium level of hydrogen sulphide production;low-to-medium parietal interaction with phenolic compounds;and non-decarboxylase activity, potentially related to the pres-ence of biogenic amines in the wine.

Thus, the selected strains could be considered as promisingfunctional starter cultures, acting at the same time as biolog-ical tools to remove OTA throughout the fermentation.

Conclusion and future trends

To our knowledge, this represents the first review regardingexclusively the biological removal of OTAmediated by yeastsor yeast-derived products in winemaking process. Based onthe available reports, toxin removal was dependent on theyeast strain or on the use of yeast-derived products. In addi-tion, several physico-chemical factors have been found toaffect OTA removal ability throughout alcoholic fermentation(i.e. temperature and sugar and nitrogen concentration) orduring the ageing of wine with yeasts or yeast cell walls (i.e.ethanol, temperature and pH). However, the toxin could bereleased back into the medium after its adsorption and this traitis strain-dependent.

Thus, ochratoxin A removal by yeasts is a promising andfriendly solution to attain the decontamination without usingharmful chemicals and without losses in nutrient value orpalatability of decontaminated food; however, the availableresults could be labeled as the first step to design practicalcommercial technologies at a larger scale. In fact, furtherintensive screening of yeasts may lead to the detection ofefficient and applicable cultures acting at the same time asstarters and as biological tools to remove OTA, with lowOTA-release ability. On the other hand, further investigationsare required, since the use of yeast-derived products or heat-and acid-treated cells could negatively affect the quality ofwine.

As a future perspective, the use of molecular biologytechniques could help design strategies for the genetic im-provement of OTA-removing yeasts, whilst the application ofpredictive models to estimate the effects of toxin concentra-tion, environmental and nutritional variables on OTA removalcould be a very useful tool for wine industry.

An open question on the link yeast/bioremediation is asfollows: Are yeasts considered as adsorbing tools or po-tential starter strains? Based on the available reports, wecan conclude that OTA levels can be reduced up to accept-able limits (2 μg l−1) in both cases; however, well-characterized starter cultures (mainly S. cerevisiae) withhigh adsorbing abilities can represent a significant andpromising frontiers goal.

Appl Microbiol Biotechnol

Acknowledgments The authors wish to thank Miss. SilvanaRendinella for her kind co-operation.

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