Environment  Health  Techniques Mycotoxins – prevention and decontamination by yeasts

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Review Mycotoxins – prevention and decontamination by yeasts Walter P. Pfliegler1,2,3, Tünde Pusztahelyi4 and István Pócsi2 1

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Department of Genetics and Applied Microbiology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary Department of Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary Postdoctoral Fellowship Programme of the Hungarian Academy of Sciences (MTA), Hungary Faculty of Agricultural and Food Sciences and Environmental Management, Central Laboratory, University of Debrecen, Debrecen, Hungary

The application of yeasts has great potential in reducing the economic damage caused by toxigenic fungi in the agriculture. Some yeasts may act as biocontrol agents inhibiting the growth of filamentous fungi. These species may also gain importance in the preservation of agricultural products and in the reduction of their mycotoxin contamination, yet the extent of mycotoxin production in the presence of biocontrol agents is relatively less understood. The application of yeasts in various technological processes may have a direct inhibitory effect on the toxin production of certain molds, which is independent of their growth suppressing effect. Furthermore, several yeast species are capable of accumulating mycotoxins from agricultural products, thereby effectively decontaminating them. Probiotic yeasts or products containing yeast cell wall are also applied to counteract mycotoxicosis in livestock. Several yeast strains are also able to degrade toxins to less-toxic or even non-toxic substances. This intensively researched field would greatly benefit from a deeper knowledge on the genetic and molecular basis of toxin degradation. Moreover, yeasts and their biotechnologically important enzymes may exhibit sensitivity to certain mycotoxins, thereby mounting a considerable problem for the biotechnological industry. It is noted that yeasts are generally regarded as safe; however, there are reports of toxin degrading species that may cause human fungal infections. The aspects of yeast–mycotoxin relations with a brief consideration of strain improvement strategies and genetic modification for improved detoxifying properties and/or mycotoxin resistance are reviewed here.

: Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: Yeast / Mycotoxin / Decontamination / Biodegradation / Biocontrol Received: November 4, 2014; accepted: January 12, 2015 DOI 10.1002/jobm.201400833

Introduction Mycotoxins are low molecular weight secondary metabolites produced by filamentous fungi that are commonly resistant to a wide spectrum of environmental factors and, therefore, undergo slow degradation. They are stabile at high temperatures and at low pH values typical of the gastric juice of animals. Another aspect is the Correspondence: Walter P. Pfliegler, Department of Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, Debrecen, Egyetem tér 1. H-4032, Hungary E-mail: walterpfl[email protected] Fax: þ3652512925 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

detection of the contamination as the different mycotoxins usually occur in low concentrations. A fungal strain may be able to produce a set of structurally different secondary metabolites like mycotoxins and synergism and antagonism among the production of the different mycotoxins also occur (e.g., [1]). The presence of the different fungal strains producing one or more type of mycotoxins is a serious human and animal health risk [2, 3] and causes substantial economic loss in food and feed industry [4]. According to The Food and Agriculture Organization’s (FAO) estimation, 25% of the world’s yearly crop production are contaminated with mycotoxins, leading to significant annual losses in

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food and feed products of estimated 1 billion metric tons. Total economic loss is caused by decreased yield, depreciated crop value, reduction in animal productivity and rise of human and animal medical, and additional costs associated with mycotoxin prevention, control, and detoxification. Thereby mycotoxins have a robust economic impact all along the food and feed industry. Not surprisingly, mycotoxin contaminations represent a worldwide problem in food, feed and bioethanol industry, therefore several methods are intensively studied to prevent contaminations and/or to decontaminate products. Microbes have a great potential to reduce the enormous economic damage caused by toxigenic fungi in agriculture and the prokaryotes are the most intensively investigated in this field, followed by yeasts and, to a smaller extent, filamentous fungi [5–7]. This review focuses mainly on different aspects of interactions between mycotoxin producing fungi/mycotoxins and yeasts (including dimorphic fungi). Some yeast species may act as biocontrol agents inhibiting the growth of mycotoxins producer filamentous fungi on crops as well as on food and feed. These species may also gain importance in the preservation of agricultural products and in the reduction of their mycotoxin contamination [8, 9]. The presence of yeasts in various technological processes may have a direct inhibitory effect on the toxin production of certain molds, which is independent of their growth suppressing effect [10, 11]. Furthermore, cell walls of several yeast species are capable of adsorbing mycotoxins from agricultural products, thereby effectively decontaminating them [12, 13]. Probiotic yeasts or products containing yeast cell wall or other additives are also applied to counteract mycotoxicosis in livestock [14]. Yeasts are also known to have further desirable characteristics as some of them are able to degrade toxins to less-toxic or even non-toxic substances [15, 16]. Moreover, there is another, relatively understudied aspect of yeast–mycotoxin interactions. Yeasts and their biotechnologically important enzymes may exhibit sensitivity to certain mycotoxins, thereby mounting a considerable problem for the biotechnological industry [17–20].

Applications of yeasts Biocontrol yeasts Bacterial, yeast, and filamentous fungal biocontrol agents applied against pre- and postharvest decay and mycotoxin contamination in agricultural products are an emerging and promising strategy to amend (or substitute) chemical treatment, improved tillage and ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

storage practices, crop rotation, and planting less susceptible cultivars in order to reduce the impact of mycotoxins in food and feed chains [21, 22]. Preharvest application of suitable (e.g., tolerant of UV-radiation, dry conditions, and high temperatures) biocontrol agents may enable early colonization and effective protection against plant pathogens and, subsequently, storage molds during postharvest conditions [23–25]. Preventing the occurrence of toxigenic fungi is the most straightforward way to prevent mycotoxin contamination. Over the past two decades, yeasts have become primary tools in biocontrol research (along bacteria) because many species show promising antagonizing properties against the common filamentous fungi that contaminate fruits, vegetables, grains, or dry cured meat products, and many of these filamentous species fall into the category of toxigenic fungi [26–28]. Such antagonistic yeasts are capable of not only reducing the economic loss caused by pre- or postharvest filamentous fungal pathogens but are also helpful in prevention of mycotoxin contamination of various products, and they are also generally considered eco-friendly. The antagonistic feature of yeasts filamentous fungi may be attributed to competition for nutrients and space, secretion of antifungal compounds, parasitism on the fungal pathogens, biofilm formation, as well as the induction and stimulation of host plant resistance like eliciting a defense response involving the production of reactive oxygen species (ROS) [26]. As the efficiency of any biocontrol system relies on specific interactions among host, pathogen, biocontrol agent and environment, detailed and multilevel studies, and trials are needed to assess the antagonistic activity and benefits of selected yeast strains in a given system. So far in most cases only the decrease of incidence and aggressiveness of pathogenic and/or spoilage fungi in the presence of antagonistic agents have been taken into consideration in numerous biocontrol trials, leaving the extent of mycotoxin production in the presence of the biocontrol agents relatively less understood. Production of a toxin depends on a great variety of environmental, epidemiological and genetic factors, and growth and, hence, growth inhibition of a toxigenic mold does not necessarily mean the concomitant reduction of the mycotoxin production. For example, Kahn et al. [29] showed that Cryptococcus isolates reduced Fusarium Head Blight severity by 50– 60% on wheat; however, deoxynivalenol (DON) toxin levels were not reduced under the same conditions. In other experimental conditions, mycotoxin production decreased parallel with growth reduction. Therefore, such biocontrol studies and trials should also consider mycotoxin levels, and not merely the growth reduction

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of the target mold, when the effectiveness of candidate biocontrol microorganisms is evaluated. A few case studies have directly linked decrease in toxigenic mold incidence and growth with decrease in mycotoxin levels in different products or in laboratory conditions. Armando et al. [30] showed that certain baker’s yeast (Saccharomyces cerevisiae) strains impaired the growth of Aspergillus carbonarius and Fusarium graminearum effectively and also inhibited the ochratoxin A (OTA), zearalenone (ZEA), and DON production under laboratory conditions. S. cerevisiae inhibited total mold incidence (Aspergillus ochraceus and Aspergillus niger) and OTA contamination on coffee [31]. Lachancea thermotolerans (formerly Kluyveromyces) reduced both the growth rate of ochratoxin-producing Aspergilli and the amount of the accumulated OTA in vitro and also on detached grape berries [32]. Some other yeasts like Hanseniaspora uvarum, Pichia anomala, and P. kluyveri produced volatile organic compounds (VOC), which prevented the growth and OTA production of A. ochraceus during coffee production [33]. Successful biocontrol of Penicillium expansum by Metschnikowia spp. on different cultivars of apple was also reported, along with an analysis of patulin concentration changes of the differently treated fruits. The data demonstrated that storage conditions and cultivars of the fruit are needed to be taken into consideration for a comprehensive analysis of the biocontrol strain [34]. On grapes, Aureobasidium pullulans significantly reduced A. carbonarius incidence, resulting in reduced OTA contamination of grape must [35]. A trial with an integrated approach to control P. expansum on apples was conducted by Lima et al. using Rhodosporidium kratochvilae and Cryptococcus laurentii together with recently developed fungicides. The application of yeasts lowered both fungicide residue levels and patulin contamination under storage conditions [36]. In a study on the biocontrol activity of the native yeast flora from dry cured ham, Debaryomyces, Candida, and Hyphopichia species were found to inhibit OTA biosynthesis. The indigenous yeasts had an antagonistic effect on the growth of Penicillium nordicum as well. H. burtonii and C. zeylanoides were the most effective both in the reduction of the growth and the OTA biosynthesis of the mold [27]. Examples of biocontrol studies considering toxin levels are listed in Supporting Information Table S1. Inhibition of mycotoxin production Yeasts may produce metabolites that have a significant suppressing effect on the expression of genes related to mycotoxin biosynthesis and/or inhibit the growth of filamentous fungi. The major VOC 2-phenylethanol (2ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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PE) produced by P. anomala, inhibited spore germination and toxin biosynthesis in the aflatoxin B1 (AFB1) producing P. flavus. Some aflatoxin biosynthesis genes like aflC (polyketide synthase, an early gene in the AF pathway), aflR (a positive aflatoxin pathway regulator), aflS (transcription enhancer), aflO (O-methyltransferase B), and aflK (versicolorin B synthase) were downregulated more than 10,000 fold after 2-PE treatment and at a concentration of 2 ml of the compound, AFB1 production was not detected in A. flavus cultures. Additionally, 2-PE also altered the expression pattern of some chromatin modifying genes (MYST1, MYST2, MYST3, gcn5, hdaA, rpdA), which influenced negatively the growth of the pathogenic mold [11]. Inhibition of the biosynthesis of OTA, which is one of the most important mycotoxins, has also been linked to various yeasts. It is notable that strains of P. anomala and S. cerevisiae were able to significantly reduce OTA production of Penicillium verrucosum, while the growth inhibition effects of these yeasts were less marked. The underlying mechanisms were not investigated in details but no adsorption or degradation of the toxin were observed, indicating that OTA biosynthesis itself was inhibited [10]. Transcriptional analysis is a useful tool to decide whether the inhibition of the toxin biosynthesis is responsible for the reduction in the toxin levels (assuming that genes in the biosynthetic pathways have been identified). For example, in addition to mycotoxin adsorption, Debaryomyces hansenii was able to inhibit OTA biosynthesis of Aspergillus westerdijkiae at the level of transcription [37]. A wine strain of S. cerevisiae was found to antagonize the growth of Aspergilli and it also inhibited OTA production of A. carbonarius and A. ochraceus through repression of the pks (polyketide synthase) biosynthetic gene [38]. Interestingly, stimulation of mycotoxin biosynthesis by yeasts has also been recorded: aflatoxin production of A. flavus was increased when co-incubated with Hyphopichia burtonii [39]. Biodegradation of mycotoxins Biodegradation is one of the most promising features of yeasts applied against mycotoxins. Mostly bacterial additives have been investigated for the decontamination and detoxification of DON, one of the most abundant and most important trichothecene toxins in food and feed, but yeasts were also proven to be useful against this toxin. Detoxification capabilities of S. cerevisiae, Geotrichum fermentans, Kluyveromyces marxianus, and Metschnikowia pulcherrima strains have been tested on wheat flour and composite fodder contaminated with

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different mycotoxins, and the DON content was significantly reduced in the samples with a greater effectiveness in the fodder [40]. However, the authors did not specify the experimental conditions and further studies are needed to evaluate trichothecene degradation and/or adsorption capabilities of these promising yeast strains. Although fumonisin B1 (FB1) is toxic to most domestic animals, and epidemiological data also suggest that exposures to fumonisins result in human esophageal cancer, the information available on the biodegradation of fumonisins by yeasts is rather limited. Out of five S. cerevisiae strains investigated by Štyriak et al. [41], merely one reduced FB1 concentration by 55% after five days of incubation via presumed biodegradation although the toxin adsorption to cells was not tested. In other studies, the black yeasts Exophiala spinifera and Rhinocladiella atrovirens were found to grow on FB1 as sole carbon source and to produce enzymes that metabolize FBs [42, 43]. Two mycotoxin degradation products arose in treatments with E. spinifera cultures, N-acetyl alkaline hydrolyzed FB1 (N-acetyl AP1) and a new compound, 2-oxo-12,16-dimethyl-3,5,10,14,15-icosanepentol hemiketal. It has to be noted that hydrolyzed fumonisin products still exhibit cytotoxicity in mammals [44]. Yeast-based biological methods for the control of OTA have also been considered for a long time as suitable alternatives to commonly used physical and chemical treatments. Similar to other microorganisms, yeasts can degrade and/or adsorb OTA and the underlying mechanisms are relatively well characterized. However, as noted in a recent review by Abrunhosa et al. [45], not all papers published so far distinguished correctly between biodegradation and adsorption. Biodegradation of OTA by yeasts have been linked to several S. cerevisiae strains [46], and also to some non-Saccharomyces yeasts such as Hanseniaspora (anamorph: Kloeckera), Trichosporon, Rhodotorula, and Cryptococcus spp. [46, 47]. Trichosporon, Rhodotorula, and Cryptococcus spp. were able to split the amide bond of the OTA molecule and release non-toxic ochratoxin a (OTa) [47]. The dimorphic species Trichosporon mycotoxinivorans was even named after its remarkable ability to detoxify OTA and ZEA [48]. However, its practical use has been questioned after T. mycotoxinivorans was recognized as a potential human pathogen [49]. OTA was also degraded successfully by the dimorphic fungus A. pullulans in grape juice and wine, again through the hydrolysis of the amide bond [50]. A strain of the astaxanthin producer yeast Phaffia rhodozyma was also able to degrade 90% of OTA in two weeks of incubation, when the mycotoxin was present at a concentration of 7.5 mg l1. OTA was converted into ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

OTa by P. rhodozyma, the adsorption of OTA by both viable and heat-treated cells was also demonstrated. The authors suggested that the biodegradation capability of the yeast was related to a metalloproteinase which was similar to carboxypeptidase A as metal-chelating agents inhibited the degradation of the mycotoxin. OTA degradation was also observed with Xanthophyllomyces dendrorhous [51]. Patulin is one of the most common mycotoxins contaminating pome fruits and derived products worldwide. Several yeast species have already been investigated concerning their interactions with patulin and patulin producing molds. Some strains of Pichia caribbica are useful in the biocontrol of several molds, important patulin-producer species among them as well. The antagonistic effect of this yeast on the growth and spore germination of Penicillium expansum under postharvest conditions was investigated recently along with its patulin degrading ability. Patulin production by P. expansum in apples was significantly reduced after incubation with P. caribbica for 15 days. In vitro tests indicated that P. caribbica degraded the mycotoxin directly [52]. Several other yeast species including M. pulcherrima, P. ohmeri, R. kratochvilovae, S. cerevisiae, and Sporobolomyces roseus have also been investigated for their patulin-degrading capabilities [53–56]. A R. kratochvilovae (a basidiomycote yeast) strain was able to convert patulin to less toxic desoxypatulinic acid (DPA), representing a novel biodegradation pathway and detoxification process [54]. A strain of the basidiomycote yeast Sporobolomyces sp. degraded patulin, and in the process, two different breakdown products were formed, desoxypatulinic acid and (Z)-ascladiol. Patulin degradation mechanisms in R. kratochvilovae and in Sporobolomyces sp. are hence different. The authors investigated the genes responsible for the toxin degradation in Sporobolomyces sp., and their results implied that patulin degradation proceeded through a multi-step process, which included the initial adaptation to patulin as a stress generating agent and followed by two separate pathways for the degradation of the toxin [56]. Patulin biodegradation capability studies carried on cider strains of S. cerevisiae suggested that under active fermentation, but not under aerobic growth, the yeast degraded the mycotoxin and the more polar products remained in the clarified fermented cider [57]. The authors identified these products as two different isomers of ascladiol, (E)-ascladiol, and (Z)-ascladiol, the toxicities of which are not well-known. Importantly, patulin did not bind to yeast cells or apple juice sediment in these model experiments. Coelho et al. [53] found that a P. ohmeri and a S. cerevisiae strain were able to detoxify patulin with

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great efficiency in both culture medium and apple juice (the mycotoxins contents were reduced by more than 99% after 5 days) but, at least partially, toxin adsorption also contributed to this achievement. On the other hand, two strains of M. pulcherrima were tested in liquid medium amended with patulin, and meanwhile yeast cells completely degraded mycotoxin within 48–72 h, patulin was not adsorbed by yeast cell wall and had no influence on yeast cell count during growth [55]. Zearalenone (ZEA) is a potent non-steroidal estrogenic mycotoxin, known as one of the most hazardous natural endocrine disrupting chemicals. ZEA is produced by several Fusarium spp. most frequently on maize and other cereal grains, causing serious problems in the food and feed industry. ZEA was eliminated from wheat flour and fodder with a promising efficacy using strains of S. cerevisiae, G. fermentans, K. marxianus, and M. pulcherrima yeasts [40]. However the study did not estimate the contribution of biodegradation- and adsorption-based processes in the reduction of the mycotoxin contents. The yeast T. mycotoxinovorans was also found to be a potent ZEA-decomposing organism, as it opens the macrocyclic ring of ZEA at the ketone group at position C60 thereby giving rise to a non-estrogenic product ZOM-1. This metabolite showed no estrogenic activity even at 1000-fold higher concentration than ZEA, and showed no interaction with the human estrogen receptor in vitro [58]. A symbiotic yeast from the cigar beetle, Symbiotaphrina kochii, was reported to grow on a diversity of mycotoxins (DON, mycophenolic acid, OTA, sterigmatocystin, ZEA) as sole carbon sources (see also Supporting Information Table S1) [59], but these interesting results could not be replicated using DON and ZEA by Karlovsky et al. [60]. Beauvericin (BEA), an antibiotic and insecticidal cyclic depsipeptide produced by Beauveriana bassiana and certain Fusarium strains and considered a minor contaminant in cereal products, was found to be very effectively degradable by incubation with S. cerevisiae strains or by the intracellular raw enzymes of these in studies conducted both with a BEA model solution and contaminated corn flour [61, 62]. Studies concerning mycotoxin biodegradation by yeast strains are summarized in Supporting Information Table S1. Detoxification by adsorption Yeast cell wall makes the cells capable to adsorb a wide array of compounds from the environment. Viable cells, non-viable cells and cell wall products of probiotic yeasts with high adsorption capacity are capable of reducing bioavailability of toxins in food and feed. Yeast species are highly diverse in cell wall composition and, not ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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surprisingly, in adsorption capacity. Yeast b-D-glucans [63], glucomannans [64] and mannan-oligosaccharide cell wall components [65] have been demonstrated to be responsible for the adsorption of mycotoxins. A standard method was recently developed to quantify the adsorption efficiency of yeast-based products (inactivated yeasts and yeast cell wall products) where the authors analyzed AFB1, OTA, and ZEA adsorption [66]. The adsorption capacity was dependent both on the yeast product and type of the mycotoxin, but it was not in direct correlation with any biochemical feature of the yeast product (e.g., mannan or glucan content). Toxin decontamination seems to require the structural integrity of the yeast cell wall [67, 68] and non-viable (e.g., heat or acid-treated) cells are generally more effective in their adsorption capacity [69–71]. Toxin removal was shown to be a very rapid process and the binding reaches a saturation relatively fast [70]. The amount of removed toxin depends both on the toxin and microorganism concentrations [72] and total amount of cell wall [73]. The b-D-glucan content and its three-dimensional arrangement in the cell wall was shown to be affecting adsorption [74]. Furthermore, it was noted that in addition to the efficiency of mycotoxin removal the kinetics of adsorption and toxin release should also be taken into consideration when oenological starter strains are selected [75]. Mycotoxin detoxifying yeasts are considered in the food and feed industry or are employed as probiotics for livestock, and beneficial effects on the organoleptic characteristics are also foreseeable when the decontaminated products, e.g., wine, are for human consumption [12]. It was demonstrated recently that the potential of wine yeasts to adsorb OTA is genetically controlled and it is a polygenic inheritable trait, which can be exploited in breeding strategies to develop more effective mycotoxin-adsorbing strains [12, 76]. Aflatoxins (AF) and sterigmatocystin (ST) are chemically related mycotoxins, and they are among the most toxic, mutagenic, and carcinogenic natural products known to date. In feed, biological decontamination of AF by microorganisms is a well-established strategy. Inclusion of lactobacilli and/or yeasts in the diet or in the fermentation procedures of feed reduces the deleterious effects of mycotoxins on farm animals by decreasing the availability and the absorption of AF in the gastrointestinal tract [77]. It is remarkable that probiotic strains of S. cerevisiae bind effectively the most carcinogenic aflatoxin B1 (AFB1) [14, 67, 78], survive gastrointestinal conditions and even improve the ruminal fermentation of feed [79]. The biosafety of aflatoxindecontaminating viable yeasts has also been studied on

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rats fed with AF-contaminated feed and, importantly, no genotoxic or cyctotoxic effects were detected, meanwhile, the reduction of the toxic effects of AF was promising [72]. The application of S. cerevisiae is also a promising strategy to reduce AFB1 contamination under storage of food, e.g., up to 55–74% of the detectable toxin content was removed after seven days of incubation in a study conducted with stored peanuts under laboratory conditions [80]. In other studies, the efficiency of mycotoxin-binding was higher (up to two-fold) when non-viable, heat- or HCl-treated cells were applied and the binding reached a saturation relatively fast [70, 81]. Considering that physical adsorption (physisorption) and desorption of AFB1 took place on the surface of the microorganisms, a physical adsorption model was proposed for the binding of AFB1 to S. cerevisiae and lactic acid bacteria [67]. The reduction of the AFB1 derivative aflatoxin M1 (AFM1) in dairy products is also possible by adsorption to S. cerevisiae polysaccharides [82]. The aflatoxin binding capabilities of yeasts and yeast products have been summarized comprehensively by Oliveira et al. [83]. Fumonisin B1 was successfully removed by a S. cerevisiae strain through physisorption as reported by Pizzolitto et al. [84]. Fumonisin-detoxifying organisms are exploitable in the agriculture and an optimization study for cost-efficient biomass production by a probiotic S. cerevisiae strain was recently published [85]. Oenological S. cerevisiae and S. bayanus strains were shown also to adsorb OTA in grape and fruit juice and in liquid culture too [86–91]. OTA removal was shown to be a very rapid process [92]. Importantly, OTA contamination was also reduced in alcoholic fermentation processes such as brewing or vinification [93]. From wine, up to 70% of OTA was removed by adsorption on yeast, and a significant percentage of the removed toxin was detectable in yeast lees [88]. When wine yeasts were tested in in vitro fermentations it was noted that in addition to the efficiency of mycotoxin removal the kinetics of adsorption and toxin release should also be taken into consideration when oenoloical starter strains are selected [75, 94, 95]. Several other yeast species including Candida spp, C. laurentii, Kloeckera spp, Rhodotorula glutinis, Saccharomycodes ludwigii, Schizosaccharomyces pombe, and Torulaspora delbrueckii [88, 96] have also been tested for their OTA removing capability, which is clearly species and strain-specific. Non-viable cells were generally shown to be more effective than alive yeasts, and the adsorption capacity was elevated after heat or acid treatments of the cells [69, 86, 96]. OTA biosorption by vinasse containing yeast cell wall, purified yeast b-glucan, and dried yeast cell wall ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

fractions was also investigated and the last two components were the most efficient OTA adsorbers [97]. Inactivated yeasts successfully reduced patulin contamination in apple juice without affecting quality parameters, which is a highly beneficial feature of this decontamination method [71, 98]. Nevertheless, the quality of wine, especially that of red wine, was more sensitive to such treatment and, therefore, strain selections should focus more on the selectivity of the mycotoxins-removing technology in this case [12]. Yeast cell wall products were also effective in ZEA adsorption [99]. Yeast cell-based feed additives, which are applied for mycotoxins-binding from the gastric content of livestock, have been tested for ZEA and AF detoxification procedures in vitro in different buffer solutions and in real gastric juice as well. However, there was no correlation between cell wall composition and the effectiveness of toxin removal. Meanwhile, the tested yeast-based additives showed very variable efficacy while other non-yeast-based products were more effective [100]. Gastrointestinal ZEA and OTA adsorption by viable S. cerevisiae cells was also investigated by Armando et al. [73] with some more promising results. The authors reached the conclusion that the relation between cell diameter and cell wall thickness correlated positively with the amount of cell wall and, consequently, with mycotoxin adsorption. The b-D-glucan content and its three-dimensional arrangement in the cell wall was the most important factor affecting ZEA adsorption [74]. It can be concluded that yeast-based products that contain cell wall components or inactivated yeasts are more widely used (and investigated) and often adsorb certain mycotoxins more effectively than live yeasts. However, the live cells have a great potential and advantage in strain improvement. They may be selected for multiple favorable traits, e.g., good fermentation capability, less marked changes in organoleptic characters of treated products, probiotic effects in feed, and toxin biodegradation. It was demonstrated recently that the potential of wine yeasts to adsorb OTA is genetically controlled and it is a polygenic inheritable trait, which can be exploited in breeding strategies to develop more effective mycotoxin-adsorbing strains [12, 76]. Trials and case studies concerning the toxin adsorbing capability of alive yeasts are summarized in Supporting Information Table S1. Mycotoxin sensitivity of yeasts The potential of various yeasts in mycotoxin detoxification has attracted wide interest from both academics and industry, but the cytotoxic effects of mycotoxins on yeasts themselves are relatively less understood

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(especially in basidiomycote yeasts that are infrequent in sensitivity studies). However, shedding light on the mechanism of toxicity of mycotoxins and mycotoxin resistance on lower eukaryotes should be useful in future strain selection and improvement procedures. Basic research carried out with well-known yeast model organisms like S. cerevisiae and the fission yeast S. pombe may also provide us with crucially important pieces of information, which may lead to a deeper understanding of the action of mycotoxins in yeasts. Importantly, the sensitivity of yeasts to certain mycotoxins may have a profound negative impact on versatile yeast-based biotechnological processes. For example, several mycotoxins like AF, OTA, ZEA, and DON may affect negatively the productivity, and yield of maize mash fermentation processes [101]. A statistically significant decrease in the final fermentation yields was observed with all mycotoxins studied except fumonisin, and the observed declination in the efficacy of the fermentation was attributed to the possible inhibitory effects of the toxins on the yeast growth and also on key enzymes of the fermentation. Growth of brewing yeasts was impaired by DON, fumonisin B1 and ZEA [102]. Ale and lager yeast strains were differently affected by culture conditions in the presence of mycotoxins. In another study, OTA did not affect the fermentation profiles of H. uvarum or S. cerevisiae in concentrations of up to 6 mg l1 [46]. In a study comparing the mycotoxin sensitivity of several yeasts and bacteria, K. marxianus, a yeast with a biotechnological potential, was highly sensitive to trichothecenes (T-2, HT-2, DAS, and DON) but its growth was not inhibited by other mycotoxins tested (AFB1, OTA, citrinin, patulin, penicillic acid, cyclopiazonic acid, penitrem A, and ZEA) [103]. In Saccharomyces, AFB1 caused standstill of the eukaryotic DNA replication machinery and thus led to cell-cycle delay [104]. The effects of the chemically related ST have not been studied in yeasts in detail although a Saccharomyces strain was susceptible to the mutagenic effect of this toxin [105]. At high concentrations of citrinin, which typically co-occurs with OTA and is toxic for the fission yeast S. pombe, a direct interaction was suggested between the toxin and the free sulfhydryl groups of plasma membrane proteins, leading to dose-dependent membrane fluidization [106]. Additionally, citrinin treatment initiated oxidative stress, induced cytotoxic processes with cell cycle arrest and also fragmentation of nuclei. An adaptation process coupled to redox-sensitive transcription factors was also observed [107]. In the case of patulin effects of high toxin concentration on the survival, morphology, fluidization of plasma membrane, and chromatin structure have also ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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been studied in S. pombe along with the generation of oxidative stress induced by the toxin [108–110]. In S. cerevisiae and Sporobolomyces, DNA damage and oxidative stress were also linked to patulin toxicity [56, 111]. It was noted that yeasts probably utilize stress response pathways common to other external stressors upon mycotoxin exposure and, if present, degradation mechanisms account for an elevated level of resistance [56]. Analysis of global transcriptome changes by DNA microarrays in S. cerevisiae cultures exposed to patulin showed that many genes involved in detoxification processes, oxidative stress response and DNA repair were highly up-regulated [112]. Moreover, a genetic correlation between patulin- and oxidative-stress-susceptible strains of S. cerevisiae was found, indicating a mechanistic similarity between patulin-induced and oxidative-stress-elicited stress responses [113]. Mechanisms underlying DON sensitivity and resistance in yeasts are less characterized. Gardiner et al. [114] found that increased cysteine or GSH supplementation resulted in higher DON resistance of a toxin-sensitive yeast strain and conjugation of the toxin to GSH was confirmed. The epoxide ring of trichothecenes are known to interact with yeast enzyme thiol groups, causing enzyme inhibition and lower yield in fermentation [17–19]. For ZEA, non-estrogenic, oxidative stress-induced effects were described in S. pombe recently as a novel aspect of ZEA toxicity. Treatment with 500 mmol l1 ZEA resulted in a 66% decrease in the glutathione concentration, accumulation of ROS, alterations in the sterol composition of the yeast, cell cycle arrest, and fragmentation of nuclei [115]. Nevertheless, effects of ZEA on yeast species currently used in mycotoxin decontamination remained to be elucidated. ZEA was also found to impair free amino nitrogen metabolism of brewing yeasts, along with OTA [116]. The effects of mycotoxins on the cellular processes and components of yeasts known hitherto are summarized in Fig. 1.

Strain improvement, future prospects The beneficial effects of yeasts interfering with the growth and mycotoxin production of toxigenic molds and/or neutralizing mycotoxins can only be harnessed when different basic and applied research approaches as well as in vitro, in vivo, and field studies are combined. Until now, most yeast-based studies carried out in this field have focused on the application of natural yeast strains because the application of transgenic organisms is highly unfavoured in the food industry by current regulations and consumer attitude. Nevertheless, some

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Figure 1. Known cytotoxic and inhibitory effects of mycotoxins on yeasts.

mycotoxin-degrading enzymes have already been heterologously expressed in transgenic yeasts with encouraging results. For example, efficient decontamination of ZEA was achieved by a genetically modified S. cerevisiae strain harboring a lactonohydrolase gene from the filamentous fungus Clonostachys rosea. The transgenic strain degraded ZEA without accumulating detectable amounts of b-zearalenol, a toxic derivative of ZEA [117]. Furthermore, an Acinetobacter-derived peroxiredoxin gene was also successfully inserted into S. cerevisiae, which oxidized ZEA to smaller, but yet estrogenic metabolites [118]. Two Fusarium trichothecene 3-Oacetyltransferases have also been cloned and expressed in baker’s yeast [119] and the transgenic strains were able to convert about 26–28% of DON during a trial bioethanol fermentation. Genetic manipulation and the use of genetically modified microorganisms (GMMs) is merely one of the options for improved detoxification when yeasts are considered. Only a minute fraction of species and strains ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

potentially useful in biocontrol, toxin adsorption, and/or degradation have so far been tested on a limited number of toxin types and conditions. Yeasts from various substrates, from toxin-contaminated substrates to arthropod guts, may be exploited against toxin producing fungi and mycotoxins. Observations on the detoxification and resistance mechanisms of known or newly isolated yeast strains may also be useful in the creation of novel GMM strains. Strain improvement may also make use of some specific characteristics of yeasts compared to bacteria: the former are capable of mating and form zygotes of two different strains even from different species, leading to the mixing of different desirable characteristics. Moreover, meiotic reproduction (spore formation) alters yeasts’ genomes and this phenomenon, along with the related genome shuffling method, may also be exploited to generate novel phenotypes with the advantage that these organisms are not regarded as GMMs. These aspects of yeasts have, however, not yet been exploited in the efforts against

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mycotoxins, but are more often investigated in the fermentation industry [120, 121]. Yeast interspecific hybrids are regarded as potentially useful in industrial applications [122, 123], especially in winemaking, where hybrids have already been selected for their distinct heterosis (hybrid vigor) effect and the usefulness of large-scale outbreeding to obtain superior industrial yeasts was demonstrated [124]. Hybrid yeasts may exhibit significantly different physiological characteristics from those of the parent species and these traits are especially prone to further changes in meiotic progeny generations (if the hybrid is not sterile) [125] and also during the course of mitotic generations. The genomes of yeasts, especially those of Saccharomyces spp. and their hybrids frequently experience substantial changes affecting chromosome number and rearrangements having a profound effect on physiological characters as well [126, 127]. Genome stabilization by breeding and selection is therefore desirable for strains applied in industry, whereas at the initial phase of strain improvement, the isolation of meiotic progenies can provide strains with diverse characteristics to be screened for. Moreover, reverse strain engineering, such as evolutionary approaches, genome shuffling, and

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sexual recombination of favorable yeasts aided by extensive screening for the improvement of desirable characteristics (biodegradation ability and speed, biocontrol properties, toxin accumulation, toxin resistance, no negative effect on product quality, etc.) could provide us with a safe and versatile alternative to the construction of genetically modified strains. So far, a study conducted on the OTA adsorbtion of S. cerevisiae strains and their meiotic descendants has already shown that even simple breeding strategies can be advantageous, quick and effective, as significant improvement was reached in the detoxification capability in the offspring generations [76]. Obviously, a more effective strain development technology would require a much deeper understanding of the molecular mechanisms underlying the sensitivity of yeasts against mycotoxins, the toxin accumulation (and release), biodegradation, unwanted effects on the quality of the detoxificated products, the transcriptional and post-transcriptional control of toxin production, and the inheritance and stability of detoxification-related traits of yeasts. Further studies are needed to estimate the applicability of conventional strain development methods and the efficiency of selection for resistance

Figure 2. Possible pipeline of strain improvement for yeasts strains applied in mycotoxin prevention and/or decontamination. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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against one or several different mycotoxins to gain highefficiency mycotoxin-decomposing strains for technological use. Future approaches may also rely on the combination of different microorganisms, both bacteria and fungi, to exploit the synergism in mycotoxinsdegradation, which possibly exist between yeast partners. Possible antagonistic effects (e.g., killer toxins of yeasts) among yeasts applied in combination and eventual pathogenicity of microorganisms [128, 129] in food industry must also be taken into account and screened for. These mean further targets for strain improvement approaches. All these efforts may help us to develop an effective pipeline of strain selection (Fig. 2), development, and industrial application (amended with technological improvement regarding mass production and formulation), aiming at the effective management of mycotoxins-contaminated food, feed and biomass. So far, a limited number of commercially available yeast products have been shown to be effective in the context of mycotoxins. Feed additives based on yeast cell wall or probiotic yeasts for livestock are practical for their toxic adsorption capabilities (as discussed in section “Detoxification by adsorption”) while some commercially available biocontrol yeasts are applied on grape, fruits and cereals in pre- or post-harvest manner against molds, including toxigenic Aspergillus, Penicillium, and Fusarium species (e.g., Candida oleophila in the products 1 1 Aspire and NEX0101 , Cryptococcus albidus in Yield 1 1 Plus , M. fructicola in Shemer ). Yet, the effect of these yeast strains on the production and accumulation of mycotoxins has not been explicitly tested. In addition to their known antagonistic effect on the incidence of plant pathogens, widespread agricultural use of promising yeast strains would potentially benefit from more emphasis on their applicability against mycotoxin contamination. Given the ever-accumulating knowledge on the biology, genetic manipulation, and biotechnological applications of yeasts and their established strain development methods, these organisms are promising counterparts of bacteria in the field of biological mycotoxin prevention and decontamination.

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Acknowledgments The authors are thankful for the anonymous reviewers for valuable comments and additions. Conflict of interest statement The authors declare no conflicts of interest.

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