Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5814-4

MINI-REVIEW

Decontamination of ochratoxin A by yeasts: possible approaches and 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 using microorganisms is one of the well-known strategies for the management of mycotoxins in foods and feeds. Yeasts are an efficient biosorbant, used in winemaking to reduce the concentration of harmful substances from the must which affect alcoholic fermentation (medium-chain fatty acids) or which affect wine quality in a negative way (ethyl phenols and sulphur products). In recent years, several studies have demonstrated the ability of yeasts to remove ochratoxin A (OTA) by live cells, cell walls and cell wall extracts, yeast lees. In spite of the physical and chemical methods applied to remove the toxin, the biological removal is considered a promising solution, since it is possible to attain the decontamination without using harmful chemicals and without losses in nutrient value or palatability of decontaminated food. In addition, adsorption is recognized as economically viable, technically feasible and socially acceptable. This paper intends to review the current achievements of OTA removal mediated by yeasts, the recent updates in the selection of strains acting at the same time as starters and as biological tools to remove OTA and the factors affecting the removal process. Keywords Yeasts . Ochratoxin A . Removal . Starter cultures . Adsorbing tools

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, Italy e-mail: [email protected] A. Bevilacqua e-mail: [email protected]

Introduction The contamination of food and feed by mycotoxins [toxic metabolites of fungi] has been recently characterized by the World Health Organization (WHO) as one of the most significant sources of food-borne illnesses (Anly and Bayram 2009); in fact, according to the Food and Agricultural Organization (FAO) of the United Nations, up to 25 % of the world’s agricultural commodities have been estimated to be significantly contaminated with mycotoxins (Jard et al. 2011). On the other hand, at least 100 countries have regulations regarding levels of mycotoxins in the food and feed industry (van Egmond et al. 2007). Currently, approximately 400 secondary metabolites with toxigenic potential produced by more than 100 moulds have been reported (Jard et al. 2011), and this number is expected to increase, probably due to the rising number of extreme weather events (Paterson and Lima 2011). Aspergillus, Penicillium and Fusarium fungal genera were considered the main sources of contamination (Reddy et al. 2010). Some mycotoxins with a strong impact on human health are 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 thermostable and can remain present in crops even after all signs of the fungus itself have been removed (Inoue et al. 2013). In recent years, OTA has received a special focus due to its toxic effects and high incidence in a wide range of foods, including cereal products, coffee, spices, beer, grape and its derivates and products of animal origin (Covarelli et al. 2012). Ochratoxin A is a nephrotoxin with immunosuppressive, teratogenic and carcinogenic properties, as stated in 1993 by the International Agency for Research on Cancer (IARC). In humans, OTA is frequently reported as the possible causative agent of an endemic kidney disease observed in the Balkans

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(Balkan endemic nephropathy and related urinary tract tumors) (Quintela et al. 2013). Ochratoxin A is continuously recovered from the blood of healthy humans, and this datum confirms the continuous exposure to the toxin (Pozo-Bayón et al. 2012); a reason for this high incidence is probably the importance of wine in the diet (ca. 13 % of the total dietary intake) (Scientific Cooperation on Questions relating to Food-SCOOP, task 3.2.7; European Commission 2002). Some authors reviewed in the past the physical, chemical and biological strategies to remove and/or decrease the content 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 tools to achieve the bioremediation of the toxin. Therefore, the aim of this paper was to specially focus on the role of yeasts as biological 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 for the first time by Zimmerli and Dick (1995, 1996), and since then, the toxin was recovered in grapes, musts and wines in several 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 Aspergillus ochraceus are considered to be the main OTA-producing species, there is strong evidence for the role of Aspergillus carbonarius in OTA contamination in grape product chain (Quintela et al. 2013). A. carbonarius is a saprophyte in the top layer of soil beneath vines and grows in berries injured by biotic agents, pests and diseases, and by abiotic factors. The grape berry moth (Lobesia botrana) is a major cause of colonization by black Aspergillus species (section Nigri), due to its active role in transporting spores into injured berries (Cubaiu 2008). The temperature, the moisture, the aeration, the period of infection and the interaction between different fungi play a significant role in mycotoxin diffusion (Esti et al. 2012). The level of OTA relies upon the types of wines and vine products, regions and vintages (Solfrizzo et al. 2010). In fact, red wines have generally higher levels than white wines, due to the increased time of contact between berry skins and grape juice during the mashing stage (Cubaiu 2008). In addition, the content of the toxin is higher in sweet and special wines, if compared to dry ones due to the different oenological practices (Covarelli et al. 2012). A survey performed by the European Commission on 1,470 samples revealed that the average value of toxin is 0.36 μg l−1, although the wines from Southern Europe suffer

a higher contamination (15.6 μg l−1). High levels of OTA were also reported on dried vine fruits (e.g. sultanas and raisins), the overall mean level being 3.10 μg kg−1 as a result of a survey on 593 samples (European Commission 2002). Based on the available scientific toxicological and exposure data, Regulation (EC) No. 123/2005 (European Commission 2005) set the limit of OTA to 2 μg l−1 for wine, grape juice, grape nectar and grape must intended for direct human consumption; however, many trade agreements require lower limits (e.g. 0.5 μg l−1) (Solfrizzo et al. 2010). On the other hand, the maximum level of OTA in dried vine fruits was set to 10 μg kg−1 (European Commission 2005).

Measures for preventing OTA appearance and possibilities of its removal Both preventive and corrective approaches were proposed to reduce the incidence of OTA contamination; the prevention strategies include the use of biocontrol agents and fungicides towards A. carbonarius and insecticides against L. botrana. However, these approaches cannot completely prevent the problem, and severe contamination of wine can occur especially for some susceptible grape varieties in certain high-risk regions or vintages with climatic conditions promoting the infection by A. carbonarius (Solfrizzo et al. 2010). Physical decontamination of OTA involves first the removal of mouldy grapes or bunches before entering in the winemaking process (Leong et al. 2006). According to Quintela et al. (2013), this method may be able to reduce OTA incidence up to 98 %, but it might not be economically feasible for the wine industry. Wine filtration through a 0.45-μm membrane reduced OTA level by 80 %; on the other hand, a filtration through a 10-μm membrane does not reduce significantly the level of the toxin (Quintela et al. 2013). Another approach to remove OTA from contaminated wine involves the use of inorganic adsorbent such as aluminosilicates, zeolites, bentonites, clays and activated carbon. A limit for their use is that they could decrease the nutritive value and organoleptic properties and increase the cost of food production (Piotrowska et al. 2013). In addition, some fining agents commonly used in winemaking process could cause adverse reactions in susceptible wine consumers (Quintela et al. 2013). Oak wood fragments can be used to reduce the levels of OTA in wine. The effectiveness of this treatment relies upon the wood format (chips or powder), quantity, time of contact and wine composition (Quintela et al. 2013). The rules for the use of this kind of approach were reported in Commission Regulation (EC) No. 1507/2006 (European Commission 2006); however, the use of oak wood fragments is forbidden in some European regions (Quintela et al. 2013). Finally,

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

Bioremediation of OTA: scientific background for 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). In addition, microbial-derived enzymes with carboxypeptidase A activity (CPA) can also degrade OTA (Amézqueta et al. 2009). However, there are still some concerns about the toxicity of the products of enzymatic degradation and the undesired effects of non-native microorganisms on wine quality (Cubaiu 2008). Another approach for the bioremediation is the adsorption of OTA on cell surface/cell surface components (Amézqueta et al. 2009). Adsorption effects were reported for Lactobacillus rhamnosus (Turbic et al. 2002), Lactobacillus spp. (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 acid bacteria (LAB) (i.e. Oenococcus oeni RM11, L. plantarum CECT 748T and L. brevis RM273) (Del Prete et al. 2007). The peptidoglycan and polysaccharides are probably involved in the 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 adsorbing properties toward OTA in grape juices and musts (Bejaoui et al. 2005). Yeasts could retain different wine compounds; thus, their use as adsorbing tools to eliminate harmful and/or negative compounds (medium-chain fatty acids, ethyl phenols and sulphur products) could be of interest (Jiménez-Moreno and Ancín-Azpilicueta 2009). Some researchers reported the idea that wine yeasts could efficiently remove OTA (Amézqueta et al. 2009); this idea was supported by five key factors: (1) it was not possible to find wine products from the degradation of OTA (Bejaoui et al. 2004; Cecchini et al. 2006), (2) acid or heat treatment retained the ability to bind the toxin (Bejaoui et al. 2004; Núñez et al. 2008), (3) the recovery of OTA in yeast lees (Caridi et al. 2006; Cecchini et al. 2006), (4) yeasts without cell wall (protoplast) were not able to adsorb the toxin (Piotrowska 2012) and (5) the fate of radiolabeled OTA during the fermentation of grape juice (Lataste et al. 2004). Generally, a surface phenomenon is the result of complex interactions (van der Walls, resonance and electrostatic forces,

and hydrogen bonding) amongst the adsorbent, adsorbate and solvent. The molecular size and the physicochemical properties of OTA as well as the physical structure of the adsorbent, including the total charge and its distribution, the size of the pores and the surface area, play a significant role in OTA-binding by adsorbent materials (Huwig et al. 2001). Why is there an interaction between OTA and the yeast cell wall? The answer relies upon the chemical traits of both of them. Ochratoxin A is a complex organic compound, consisting of chlorine-containing dehydroisocoumarin linked through the 7-carboxyl group to 1-β-phenylalanine; phenol and carboxyl are the main functional groups involved in some different adsorption mechanisms. First, OTA is considered with zearalenone as the less polar mycotoxin and could then be bound on hydrophobic surfaces through the phenol group and via interactions of two-π-electron orbital. Moreover, the pKa of the carboxyl group of phenylalanine moiety is 4.4; thus, the toxin is partially dissociated at the pH of wine and carries a positive charge on the amine function (NH3+) (Leong et al. 2006). Yeast biomass may be regarded as a good source of adsorbent material, due to the presence in the cell wall of some specific macromolecules, such as mannoproteins and β-D-glucans (Ringot et al. 2007). For example, the cell wall of Saccharomyces consists of two layers: an inner layer made of β-1,3-glucan and chitin, which represents about 50–60 % of the cell wall dry weight, and an outer layer, which consists of β-1,6-glucan and heavily glycosylated mannoproteins. The mannoproteins are glycoproteins having carbohydrate fractions made of around 98 % mannose and 2 % glucose; they are covalently linked to the inner cell wall layer, either directly to the β-1,3-glucan matrix or indirectly via a β-1,6-glucan branch (GonzalezRamos and Gonzalez 2006). This structure is highly dynamic and strain-dependent, since about 1,200 genes drive the synthesis of these cell wall components. Culture conditions, including pH, temperature, oxygenation rate, kind of medium and concentration or nature of the carbon source, strongly modulate the quantity and structural properties of β-D-glucans, mannans and chitin in cell walls; moreover, the cell cycle stage also interacts with the cell wall composition. For example, budding induces strong changes in the distribution of the structural components of the cell wall such as chitin (Jouany et al. 2005). Despite the strong evidence of OTA adsorption to cell walls (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 suggested the existence of a possible pathway for the degradation of the toxin, different from the mechanism involving L -β-

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

Application of yeasts or yeast-derived products as adsorbing tools to remove OTA from synthetic and natural grape juices, wines or model wine systems Some studies focused on the reduction of OTA in synthetic and natural grape juices, wines or model wine systems by yeasts or inactive dry yeast (IDY) preparations (i.e. inactive yeast, yeast autolysates, yeast extracts and yeast hulls or walls) (Pozo-Bayón et al. 2009); Table 1 reports a short overview of the available references. The experiments were carried out under laboratory conditions, and viability was not a prerequisite to achieve the removal of the toxin. Indeed, yeasts were intended simply as adsorbing tools. The first comprehensive report on the use of yeasts and yeast-derived products intended as winemaking tools for the removal of OTA was conducted by Bejaoui et al. (2004). They reported that heat- and acid-treated cells were able to bind significantly higher levels of OTA than the viable ones in a synthetic grape juice medium. Viable yeasts bound up to 35 % of OTA, depending on yeast concentration, whilst heat- and acid-treated cells decreased the toxin by 90.80 and 73 %, respectively. In addition, a comparative experiment between heat-treated cells and commercial yeast walls, at two different concentrations (0.2 and 6.7 g l−1) in a contaminated grape juice, showed the highest efficiency by heat-treated cells able to completely remove the toxin in 5 min. This trend could be due to the fact that heating might cause changes in the surface properties of cells, like the denaturation of proteins or the formation of Maillard reaction products. On the other hand, acidic conditions could affect polysaccharides by releasing monomers, which are further fragmented into aldehydes after the breaking of glycosidic bonds. These released products could harbour higher adsorption sites than viable cells with an enhancement of OTA removal (Piotrowska et al. 2013). Several winemaking practices involve a prolonged contact between yeasts or IDY preparations and wine (i.e. ageing on lees and the production of sparkling wines), thus suggesting that yeast cells could play a significant role in OTA removal also at the end of the fermentation process (Núñez et al. 2006). During this period, some genes encoding proteins involved in cell wall biosynthesis are strongly up-regulated (fks1, gsc2, ssd1 and mpt5) (Pradelles et al. 2008); in addition, variable amounts of mannoproteins and glucans can be released into wines due to yeast autolysis interacting with wine phenolic compounds, decreasing their astringency and/or acting as protective colloids, and enhancing the color stability of red wines (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 focused on the use of vinification on lees (used successfully with white wines) for red wines (Garcia-Moruno et al. 2005); Garcia-Moruno et al. (2005) reported that OTA removal was different using white and red lees wine. After 7 days of contact (20 g l−1 of lees), the white lees wine reduced OTA by 70.90 % whilst the red lees wine decreased the toxin by 51.50 %. After 80 days. OTA reduction was ca. 73.90 % with the white lees and 63.10 % with the red ones. This difference was attributed to competition on the binding sites between OTA and polyphenols, and this hypothesis was supported by Meca et al. (2010), who reported that phenols, proteins, organic acid and colloidal particles showed the same ability of OTA to react with different compounds of the cell wall. Another trend is the use of commercial yeast derivative preparations, as an alternative to lees; these products could reduce the time required to obtain wines with physicochemical and sensory characteristics similar to those aged on lees (Del Barrio-Galán et al. 2012). Piotrowska et al. (2013) added a thermally inactivated biomass of Saccharomyces cerevisiae yeast to wines from white grape and blackcurrant juices and decreased the content of OTA by ca. 60 %. The heat treatment (85 °C for 10 min) was also proposed by Núñez et al. (2008) to increase the removal yield of whole yeast cells and yeast cell walls in a model system up to 95 %. According to Piotrowska et al. (2013), the use of inactivated yeasts as OTA-adsorbing tools might be highly advantageous since such biomass does not change organoleptic features of end products; on the other hand, Núñez et al. (2008) reported that heat- and acid-treated cells could negatively affect the quality of wine. Nevertheless, the existence of potential unexpected effects related to odorant compounds initially present in the IDY preparations (Pozo-Bayón et al. 2009) should be carefully evaluated. Since the natural autolysis is a long-lasting process, it is usual to use model systems to obtain results in shorter periods of time (Martínez-Rodríguez et al. 2001); Petruzzi et 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. As pH, temperature and ethanol are the major factors affecting autolysis 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 removed the toxin by 3.69–81.87 %; S. cerevisiae Y28 showed the higher removing percentages (24.60–81.87 %), whereas the strain RC212 removed up to 25 % of the toxin. On the other hand, yeast cell walls reduced OTA by 2.47–50.00 %. The decrease of the toxin was higher at 30 °C and pH 3.0 and

Appl Microbiol Biotechnol Table 1 Review of wine yeasts and yeast-derived products used as adsorbing tools to remove ochratoxin A (OTA) from synthetic and natural grape juices, wines or model wine systems Yeast or yeastderived product

State of cells

Type of assay

Experimental conditions

Incubation at 25 °C

Yeast cell walls

Not treated

Red wine

Saccharomyces cerevisiae

Viable

Synthetic grape juice Incubation for 2 h, 30 °C

Yeast walls additive Saccharomyces cerevisiae

Saccharomyces cerevisiae

1.9

12–30

Silva et al. (2003)

2

17.00–35.00

Bejaoui et al. (2004)

75.00–90.80

Heat-treated

Synthetic grape juice Incubation for 2 h, 30 °C

6.7–27.4

2

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

Not treated

Red juice

Incubation for 2 h, 30 °C

0.2–6.7

10

100

IY

Red wine

1–4

3.04–3.30 25–60.30

20

1.65–7.09 70.90–73.90

20

2.49–4.12 51.50–63.10

Rehydrated IY

Model wine

Stirring for the first 90 min and then stored for 7 days at ca. 20 °C without further shaking Stirring for the first 90 min and then stored for 7 or 80 days at ca. 20 °C without further shaking Stirring for the first 90 min and then stored for 7 or 80 days at ca. 20 °C without further shaking Incubation for 2 days, at ca. 22 °C Stirring for 90 min and then stored for 30 or 90 min at ca. 20 °C without further shaking Stirring for the first 90 min and then stored for 7 or 80 days at ca. 20 °C without further shaking Incubation for 214 h

Rehydrated IY and heat-treated Not treated

Model wine

Incubation for 214 h

Model wine

Incubation for 214 h

1

10

18.8

Heat-treated

Model wine

Incubation for 214 h

1

10

95.0

Lees from white Red wine must fermentation

Red wine

Yeast hulls

Not treated

Red wine

NI

IY

Red wine

IY

Red wine

Yeast cell walls

0.5–1.0 6.7–27.4

Acid-treated

Lees from red must fermentation

Saccharomyces cerevisiae

Adsorbent OTA level OTA removal References (μg l−1) (%) amount (g l−1)

2.00–5.00 5

Garcia-Moruno et al. (2005)

28.00–43.00

Leong et al. (2006)

1–4

2.55–2.78 20.00–46.50

Savino et al. (2006)

4

2.84

62.00–81.00

1

10

0.8

1

10

95.4

Candida spp., Kloeckera spp., Rhodotorula glutinis, Cryptococcus laurentii Saccharomyces cerevisiae

Biomass from culture broth Biomass thermally inactivated

White wine

Incubation for 4 h, 25 °C

NI

10

4.75–21.40

White wine

Incubation for 4 h, 25 °C

NI

10

8.08–30.45

Biomass from culture broth

Model wine

10

2

3.69–81.87

Yeast cell walls

Not treated

Model wine

10

2

2.47–50.00

Saccharomyces cerevisiae

Biomass thermally inactivated

White wine Red wine

Incubation for 9 days at 25 or 30 °C, with ethanol content of 5, 10 or 15 %, at pH 3.0 or 3.5 Incubation for 9 days at 25 or 30 °C, with ethanol content of 5, 10 or 15 %, at pH 3.0 or 3.5 Incubation for 24 h, 30 °C Incubation for 24 h, 30 °C

0.005 0.005

1,000 1,000

64.40 62.40

Núñez et al. (2008)

Var et al. (2009)

Petruzzi et al. (2014a)

Piotrowska et al. (2013)

IY inactive yeast, NI not indicated

with 15 % of ethanol. The viable count of yeasts was below the detection limit after 3 days in all the samples, thus suggesting that the bioremediation was not strictly related to cell viability.

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

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to pH 3.5, containing 10 % of ethanol and incubated at 37 °C. Based on available data, we could suppose that some variables play a major role in OTA removal efficiency of yeast derivatives, i.e.: &

&

&

The enzymatic and chemical protocol for their production, as the enzymatic and thermal procedure currently employed for their production could have a strong influence on their physical and compositional characteristics (Núñez et al. 2006); The amount (Silva et al. 2003; Bejaoui et al. 2004), although most of the researchers performed the experiments using amounts of yeast cell wall preparations of up to 0.40 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 production of autolysates, as the content of mannoproteins and glucans is usually higher in the lysates from viable cells than from rehydrated cells (Guilloux-Benatier and Chassagne 2003).

Application of yeasts to remove OTA throughout alcoholic fermentation and factors affecting its removal Some basic requirements for an ideal method to remove OTA are the low cost, the simplicity of use and the absence of effects on the quality of wine (Amézqueta et al. 2009). Unfortunately, inactivated cells or IDY products could negatively affect the quality of wine (Núñez et al. 2008; PozoBayón et al. 2009), whilst the use of viable cells does not show this drawback; moreover, active yeasts have a long history of safe use as starter cultures (Abrunhosa et al. 2010). Over the last years, some authors identified yeasts from Saccharomyces and non-Saccharomyces genera able to decrease OTA content by 0.60–100 % throughout fermentation (Table 2). Some key factors able to influence this phenomenon are the conditions for the assay (laboratory or industrial scale, in vitro or in vivo conditions), kind of the strain, cell dimension, flocculence, cell sedimentation kinetics and toxin concentration (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 especially during alcoholic fermentation, as a result of yeast activity and/ or the interaction between OTA and grape constituents (Paster 2008). In red vinification, most of OTA is removed in the

solid–liquid separation stages, when the wine or the juice is separated from the skins (Bizaj et al. 2009). In white vinification, grapes are pressed before fermentation and OTA might bind more effectively to the grape proteins and solids, which are 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 between OTA and the natural microbiota of grape must, probably contributing to the modification of the removal yield of inoculated starter strains. Differences in OTA removal may be related to the scale of assay, as the management of the fermentation could be difficult in macro-scale experiments; on the other hand, changes in OTA content during small-scale experiments might be simply managed. Under laboratory conditions (from 10 ml to 2 l), OTA was removed by 32.6–90.95 %, depending on the strain and the medium (Caridi et al. 2006; Meca et al. 2010; Cecchini et al. 2006; Esti et al. 2012). OTA removal was also found in higher volumes. Fernandes et al. (2007) reported that the toxin was reduced by 55.5– 77.7 % in microvinification trials consisting of 20 kg of grapes; however, the mass balance performed in each vinification trial revealed that this decrease was due predominantly by the adsorption onto suspended solids in musts and wines. Ponsone et al. (2009) showed that a significant reduction of OTA (i.e. 77–100 %) after fermentation in 100-l tanks was caused by an extensive adsorption onto the solid parts of the grapes and yeast lees. Only one study was carried out at industrial scale (800-hl tanks) by Grazioli et al. (2006); they reported that maceration increased OTA content by 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 strains of S. cerevisiae: Malaga LOCK 0173, able to remove OTA (1,000 μg l−1) by 82.80, 10.70 and 35.40 %, respectively, from white juice, blackcurrant juice and synthetic medium, and the strain Syrena LOCK 0201 respectively reducing toxin concentration by 85.10, 65.20 and 21.00 %. Csutorás et al. (2013) reported that a commercial S. cerevisiae type “Fermol Premier Cru” removed OTA (initial content 4,000 μg l−1) by 90, 85 and 73 % from red, rose and white must, respectively. Chemico-physical factors acting on OTA removal Although many papers focused on the role of some physicochemical factors on the course of wine fermentation (Bely et al. 2003, 2008; Hernández-Orte et al. 2006; Torija et al. 2003), few data are available on their influence on the removal of OTA by yeasts. This issue is of great concern considering that some oenological parameters can modify the surface properties of cell wall (Vasserot et al. 1997); thus, they could

20

1

1

4

1

7

7

1

1

Saccharomyces cerevisiae, Saccharomyces cerevisiae× Saccharomyces bayanus, Saccharomyces bayanus, Kloeckera apiculata, Torulaspora delbrueckii, Schizosaccharomyces pombe, Candida pulcherima, Saccharomycodes ludwigii Saccharomyces cerevisiae

Saccharomyces cerevisiae

Kloechera apiculata

Saccharomyces cerevisiae

Kloechera apiculata

Saccharomyces cerevisiae

Saccharomyces cerevisiae

0.25–18.52 0.20–6.00 0.20–6.00

T=25 °C, S=200 g l−1 T=25 °C, S=200 g l−1 T=25 °C, S=200 g l−1 Laboratory scale Laboratory scale

Rose must

Microvinification

Laboratory scale

Laboratory scale

Laboratory scale

T=25 °C

T=25 °C

T=18±1 °C

0.25–18.52

T=25 °C, S=200 g l− 1

Laboratory scale

0.88

T=20–32 °C

Industrial-scale vinification

0.41

0.41

0.43–7.48

2

T=20 °C, S=20.79°Brix

Laboratory scale

2

7.63

T=20 °C, S=22.28°Brix

T=25 °C

Laboratory scale

1.58

4.0

2

OTA level (μg l−1)

Laboratory scale

T=25 °C

T=25 °C

Microvinification

Laboratory scale

NI

Fermentative conditions

Laboratory scale

Scale of assay

Red must

Red must from Vinhão

Synthetic medium

Synthetic medium

Synthetic medium

Red must from Negroamaro and Primitivo Synthetic medium

Red must from Tinta Roriz, Touriga Nacional, Touriga Franca, Tinta Barroca and Tinto Cão White must naturally contaminated with OTA White must added with OTA White must from Trebbiano Toscano and Malvasia del Lazio Red must from Primitivo

1

Saccharomyces sensu stricto

White and red musts

1

Saccharomyces uvarum, Saccharomyces cerevisiae, Saccharomyces bayanus, Kloeckera apiculata, Torulaspora delbrueckii, Schizosaccharomyces pombe, Candida pulcherima, Saccharomycodes ludwigii NI

Type of assay

Number of strains

Yeasts

Table 2 Review of wine yeasts able to remove ochratoxin A (OTA) throughout alcoholic fermentation

44

41

55.50–77.70

25–64

28–100

32–49

28.00–48.00

30

53.21–70.13

46.83–52.16

67.89–83.34

39.81–90.95

90

40.10–72.12

OTA removal (%)

Ethanol production

Ethanol production

/

Ethanol production

Ethanol production

Ethanol production

Ethanol production

/

/

/

Ethanol production

Ethanol production

/

/

Oenological traits of yeasts

Lasram et al. (2008)

Fernandes et al. (2007)

Angioni et al. (2007)

Orro et al. (2006)

Grazioli et al. (2006)

Cecchini et al. (2006)

Caridi et al. (2006)

Ratola et al. (2005)

Morassut et al. (2004)

References

Appl Microbiol Biotechnol

1 1

Saccharomyces cerevisiae

1

1

2

1

2

Saccharomyces cerevisiae

Saccharomyces cerevisiae

Saccharomyces cerevisiae

Saccharomyces cerevisiae

Saccharomyces cerevisiae

Saccharomyces cerevisiae

1

Saccharomyces cerevisiae

2

Saccharomyces cerevisiae

16

3

Saccharomyces cerevisiae

48

1

Saccharomyces cerevisiae

Saccharomyces cerevisiae

1

Saccharomyces cerevisiae

Saccharomyces cerevisiae

Number of strains

Yeasts

Table 2 (continued)

Microvinification Microvinification Microvinification

Red must

Synthetic medium

Synthetic medium

Laboratory scale

Laboratory scale

Laboratory scale

Laboratory scale

Blackcurrant juice Synthetic medium

Laboratory scale

White juice

Laboratory scale

Blackcurrant juice Laboratory scale

Laboratory scale

White juice Synthetic medium

Laboratory scale

Synthetic medium

T=25 or 30 °C, S=200 or 250 g l−1, N=250 or 310 mg l−1

T=25 or 30 °C¸ S=200 or 250 g l−1, N=250 or 310 mg l−1

T=25 or 30 °C, S=200 or 250 g l−1

T=30 °C, S=12°Brix

T=30 °C, S=12°Brix

65.20 5.41–49.58

6.00–25.66

2.82–27.53

2

2

2

85.10

21.00

10.70

82.80

35.40

88.00–90.00

73.00–76.00

83.00–86.00

67.00

79.00

62

7.80-81.95

32.60–50.40

77–100

83–86

>40

2.00

2.60

OTA removal (%)

1,000

1,000

1,000 1,000

T=30 °C, S=12°Brix

1,000

T=30 °C¸ S=20 g l−1

T=30 °C, S=12°Brix

10–4,000 1,000

T=12 °C

10–4,000

10–4,000

15

15

15

4.10

10

0.3

6

T=30 °C, S=20 g l−1

T=12 °C

T=12 °C

T=20 °C, S=20.5°Brix

Laboratory scale

White must

T=20 °C, S=22°Brix

T=20 °C, S=20.5°Brix

T=25 °C

T=25 °C, S=22°Brix

T=24–28 °C, S= 21.50°Brix

T=24–28 °C, S= 24.70°Brix

10

3.48

T=20 °C, S=180 g l−1

T=16–18 °C, S=38°Brix

4.95

OTA level (μg l−1)

T=20 °C, S=180 g l−1

Fermentative conditions

Laboratory scale

Laboratory scale

Laboratory scale

White must from Sauvignon blanc Red must from Cesanese di Affile White must from Sauvignon blanc Rose must

White must from Zibibbo

Laboratory scale

Microvinification

Red must from Bonarda

White must from Moscato

Microvinification

Laboratory scale

Laboratory scale

Laboratory scale

Scale of assay

White must from Moscato of Saracena Red must from Tempranilo

Synthetic medium

Synthetic medium

Type of assay

Ethanol, glycerol and acids production Ethanol, glycerol and acids production Sugar consumption, ethanol, glycerol and volatile acidity production Sugar consumption, ethanol and glycerol production; phenotypical traits Sugar consumption, ethanol and glycerol

/

/

/

/

/

/

/

/

/

/

/

Sugar consumption, ethanol and acids production Sugar consumption, ethanol and acids production /

Sugar consumption and volatile acidity production Sugar consumption and volatile acidity production /

Oenological traits of yeasts

Petruzzi et al. (2014c)

Petruzzi et al. (2014b)

Bevilacqua et al. (2014)

Piotrowska et al. (2013)

Csutorás et al. (2013)

Esti et al. (2012)

Caridi et al. (2012)

Meca et al. (2010)

Ponsone et al. (2009)

Blaiotta et al. (2009)

Bizaj et al. (2009)

References

Appl Microbiol Biotechnol

NI not indicated, / none oenological trait assessed¸ T temperature, S sugar content, N nitrogen content as yeast assimilable nitrogen (YAN)

Phenotypical traits include the following: resistance to single and combined stress conditions, enzymatic activities, hydrogen sulphide production, interaction with phenolic compounds and biogenic amines formation; 1 °Brix means 1 weight percentage of reducing sugars

Petruzzi et al. (2014d) 0.60–42.80 4 Saccharomyces cerevisiae

Synthetic medium

Laboratory scale

T=25 or 30 °C, S=200 or 250 g l−1

2

production; phenotypical traits Sugar consumption, ethanol, glycerol and volatile acidity production; phenotypical traits

OTA removal (%) Number of strains Yeasts

Table 2 (continued)

Type of assay

Scale of assay

Fermentative conditions

OTA level (μg l−1)

Oenological traits of yeasts

References

Appl Microbiol Biotechnol

affect the binding capacity of yeasts. Petruzzi et al. (2014b) studied the kinetics of OTA removal by a potential starter strain (i.e. S. cerevisiae W13) as affected by the most important environmental parameter (e.g. temperature) (Pizarro et al. 2008), as well as by two major nutrients acting on the fate of must fermentation (sugar and nitrogen) (Heinisch and Rodicio 2009). At this scope, laboratory-scale fermentations were carried out at two temperatures (25 and 30 °C) and sugar levels (200 and 250 g l−1), with or without supplementation of medium with diammonium phosphate (DAP) (300 mg l−1). The yeast was able to reduce OTA up to 57.21 %, with a positive effect of temperature and sugar. These results confirmed the data by Patharajan et al. (2011), who studied six different temperatures (10, 15, 20, 25, 30 and 35 °C) to optimize OTA removal in a liquid medium by three yeasts (Metschnikowia pulcherrima MACH1, Pichia guilliermondii M8, Rhodococcus erythropolis AR14) and found the highest removal at 30 °C. Petruzzi et al. (2014b) suggested that the major ability to remove OTA at 30 °C could be associated to the release of cell wall polysaccharides. The effects of sugar concentration could be related to the level of mannoproteins, as these compounds are strictly dependent on sugar (Meca et al. 2010). Finally, Petruzzi et al. (2014b) found that the removal was highest after 3 days of fermentation; then, the toxin was partially released in the medium and the performances of the strain decreased (i.e. 6.0–25.66 % reduction). A release of toxins after their removal was also recovered for other compounds; Peltonen et al. (2001) reported the release of aflatoxin B1 (AFB1) by lactobacilli (i.e. L. amylovorus and L. rhamnosus). They suggested that this phenomenon could be associated to the weak bond in lactobacilli/AFB1 complexes. Since hydrogen bonding and ionic or hydrophobic interaction seem to be related to OTA adsorption mechanism by S. cerevisiae (Cecchini et al. 2006), Petruzzi et al. (2014b) considered this statement as a possible explanation for OTA release by yeasts. In another study, Petruzzi et al. (2014c) tested two wild S. cerevisiae strains (W28 and W46) under the same experimental conditions reported for the strain W13. As a result, the strains W28 and W46 were able to decrease the toxin by ca. 70 %, with the highest removing effect observed after 3 days at 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 medium and the performances of the strains decreased (i.e. 2.82– 27.53 % reduction). As reported above, sugar and temperature played a major role, with a significant effect of DAP supplementation, too. As regards nitrogen, we could suppose that the strains exhibited a significant ability to remove OTA only when the conditions were closer to the optimal ones (i.e. greater YAN content in the must), probably due to a higher release of cell wall polysaccharides (Giovani et al. 2010). The different trends of the three strains (i.e. W13, W28 and W46) toward DAP addition could be due to the strong strain

Appl Microbiol Biotechnol

dependence on the response to changes in environmental conditions (Giovani et al. 2010). OTA removal as a new trait to select functional yeast starter cultures Recently, a new approach for the study of OTA-removing yeasts was proposed by Petruzzi et al. (2014b, c, d), i.e. yeast strains intended as functional starter cultures, as they show both the benefits of traditional starters and a health- or product-focused function. In this sense, a drawback in the literature is that few data are available on the oenological traits of OTA-removing yeasts (see Table 2), whilst this aspect is of great concern for the possible selection of functional strains acting at the same time as starters and as biological tools to remove the toxin. Thus, the removal of OTA could be an interesting and attractive trait to complement the classical oenological characterization based on technological and qualitative key traits, such as the tolerance and high ethanol production, exhaustion of sugars, growth at high sugar concentration, good glycerol production, growth at high temperatures, low hydrogen sulphide and volatile acidity production, resistance to sulphur dioxide and good enzymatic profile (Nikolaou et al. 2006). This idea is supported by the fact that the removal of toxin by yeasts is genetically controlled and is a polygenic inheritable trait of wine yeasts (Caridi et al. 2012). However, the toxin could be released back into the medium after its adsorption (Bevilacqua et al. 2014; Petruzzi et al. 2013, 2014b, c), probably due to the nature of the adsorption mechanism involved (hydrogen bonding, ionic or hydrophobic interaction) (Cecchini et al. 2006). Strain dependence could play a significant role also on the stability of the complex cell/toxin, as well as on the yeast cell wall composition in response to a variety of fermentative/growth conditions (Bevilacqua et al. 2014). Therefore, Petruzzi et al. (2014d) used a polyphasic approach, consisting of the genotypic identification and the evaluation of the phenotypic traits and fermentative performances in a model system (temperature, 25 and 30 °C; sugar level, 200 and 250 g l−1), to select wine starters of S. cerevisiae from 30 autochtonous isolates from Uva di Troia cv., a red wine grape variety of the Apulian region (Southern Italy). The ability to remove OTA was used by the authors as a desirable functional trait to improve the safety of wine. As a result, 11 biotypes were identified and a strain, representative of each biotype, was further studied. Four strains (Y20, W21, W40 and W41) were able to reduce OTA by 0.60–42.80 %; in addition, the strains W21 and W40 were promising in terms of ethanol, glycerol and volatile acidity production, as well as for their enzymatic and stress resistance characteristics. In this sense, interesting traits were also possessed by S. cerevisiae strains W13 (Petruzzi et al. 2014b), W28 and W46 (Petruzzi

et al. 2014c), e.g. high tolerance to single and combined stress conditions; β-D-glucosidase, pectolytic and xylanase activities; low-to-medium level of hydrogen sulphide production; low-to-medium parietal interaction with phenolic compounds; and non-decarboxylase activity, potentially related to the presence of biogenic amines in the wine. Thus, the selected strains could be considered as promising functional starter cultures, acting at the same time as biological tools to remove OTA throughout the fermentation.

Conclusion and future trends To our knowledge, this represents the first review regarding exclusively the biological removal of OTA mediated by yeasts or yeast-derived products in winemaking process. Based on the available reports, toxin removal was dependent on the yeast strain or on the use of yeast-derived products. In addition, several physico-chemical factors have been found to affect OTA removal ability throughout alcoholic fermentation (i.e. temperature and sugar and nitrogen concentration) or during the ageing of wine with yeasts or yeast cell walls (i.e. ethanol, temperature and pH). However, the toxin could be released back into the medium after its adsorption and this trait is strain-dependent. Thus, ochratoxin A removal by yeasts is a promising and friendly solution to attain the decontamination without using harmful chemicals and without losses in nutrient value or palatability of decontaminated food; however, the available results could be labeled as the first step to design practical commercial technologies at a larger scale. In fact, further intensive screening of yeasts may lead to the detection of efficient and applicable cultures acting at the same time as starters and as biological tools to remove OTA, with low OTA-release ability. On the other hand, further investigations are required, since the use of yeast-derived products or heatand acid-treated cells could negatively affect the quality of wine. As a future perspective, the use of molecular biology techniques could help design strategies for the genetic improvement of OTA-removing yeasts, whilst the application of predictive models to estimate the effects of toxin concentration, environmental and nutritional variables on OTA removal could be a very useful tool for wine industry. An open question on the link yeast/bioremediation is as follows: Are yeasts considered as adsorbing tools or potential starter strains? Based on the available reports, we can conclude that OTA levels can be reduced up to acceptable limits (2 μg l −1 ) in both cases; however, wellcharacterized starter cultures (mainly S. cerevisiae) with high adsorbing abilities can represent a significant and promising frontiers goal.

Appl Microbiol Biotechnol Acknowledgments The authors wish to thank Miss. Silvana Rendinella for her kind co-operation.

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