Microorganisms and their enzymes for detoxifying mycotoxins posing

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Chapter 8

Microorganisms and their enzymes for detoxifying mycotoxins posing a risk to livestock animals I. Rodrigues1, E. M. Binder2 and, G. Schatzmayr3* 1

BIOMIN Holding GmbH, Industriestrasse 21, 3130 Herzogenburg, Austria 2 ERBER AG, Technopark 1, 3430 Tulln, Austria 3 BIOMIN Research Center, Technopark 1, 3430 Tulln, Austria

Occurrence of mycotoxins is ubiquitous. Even with the use of prevention techniques, it is virtually impossible to avoid their presence in agricultural commodities. The toxicity of these fungal metabolites brings serious risks upon humans and animals. Mycotoxicoses are animal or human diseases caused by mycotoxin ingestion, inhalation or skin-contact. In animals, these range from immunosuppression and performance effects to hepatotoxic, nephrotoxic, neurotoxic, dermal, carcinogenic, reproductive, teratogenic and gastro-intestinal effects depending on animal-, environmental- and toxin-related factors. A suitable mycotoxin risk management should take the different chemical structures of mycotoxins into consideration as a successful strategy for one mycotoxin, may fail in the elimination of another. Biotransformation and biodegradation are mycotoxin-specific methods which rely in microorganisms and enzymes’ capacity of metabolization or degradation of mycotoxins into less or non-toxic metabolites prior to their resorption in the gastro-intestinal tract. Some microorganisms have shown biotransformation capacity both in vitro and in vivo, representing effective mycotoxin risk management tools in animal feed.

Although mycotoxin-related problems had been already described in medieval times, when people affected by ergot-alkaloids exhibited swollen © 2009 American Chemical Society In Mycotoxin Prevention and Control in Agriculture; Appell, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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108 members with burning sensations, with subsequent necrosis and loss of the extremities, modern mycotoxicology begun quite recently with the discovery of aflatoxins in the early 1960s (1). Turkey-X disease was responsible for the death of more than 100 000 young turkeys in poultry farms in England and followed the consumption of groundnuts infected with Aspergillus flavus. Ever since then, the impact of mycotoxins in human and animal health has been the focus of many scientific studies and several reports are available concerning prevention, decontamination and minimization of mycotoxins risk (2, 3). Unlike primary metabolites, which are essential for fungi growth, mycotoxins are secondary metabolites produced by filamentous fungi (moulds) in the final stages of exponential growth phase (4). These chemical compounds are toxic to vertebrates upon ingestion, inhalation and/or dermal contact. Reports by Hawksworth (5,6) describe the existence of more than 70,000 species of fungi; however, this number might represent only 5% of the world’s total fungal species, estimated to be 1.5 million. Nevertheless, the majority of the known toxigenic species falls into three recognized genera: Aspergillus, Penicillium and Fusarium (1). Mycotoxin contamination often begins in the field and continues throughout harvest, transportation and storage, depending on the activity and colonization levels of fungi which are in turn determined by the prevailing environmental conditions and the nutritional components of the food matrix (7). In general, fungi are divided in two main groups, field fungi and storage fungi, depending if they occur more frequently on the field or after harvest, respectively. However, even if this terminology has been commonly used, conditions for growth of a specific organism can occur in either the field or during storage (8), especially because even the same fungal genus contains species that differ greatly in their optimum temperature for growth and for their parasitic abilities. Important mycotoxins produced by Aspergillus fungi include aflatoxin B1, B2, G1 and G2 and ochratoxin A. The latter can also be produced by species belonging to the Penicillium genus. Fusarium mycotoxins commonly impacting the health and productivity of animals are type-A (T-2 toxin, HT-2 toxin and diacetoxyscirpenol (DAS)) and type-B (deoxynivalenol (DON) and nivalenol) trichothecenes, zearalenone (ZON) and fumonisins. Reports on the worldwide occurrence of mycotoxins in commodities, feed and feed ingredients are available (9, 10, 11). Mycotoxins occurrence is ubiquitous, not only geographically but also in terms of commodities. European samples for example are more frequently contaminated with DON, ZON and T2 whereas materials from Asia and the Pacific tend to be contaminated with DON, ZON, fumonisins and aflatoxins (9). Nevertheless, agricultural trade globalization and climate change might also have a role on the contamination pattern, leading to the occurrence of mycotoxins in regions where originally they would not be found at. Approaches to prevent mycotoxicoses include pre-, harvest and postharvest strategies. The latter comprise physical, chemical, biological methods and use of adsorbents. Pre-harvest strategies have been fastidiously reviewed by Jouany (2) and will not be object of discussion in this paper. Furthermore, preharvest control of mould growth is greatly compromised by the inability of man to control climate, a critical factor on mould contamination and mycotoxin production. Physical and chemical treatments of contaminated grains

In Mycotoxin Prevention and Control in Agriculture; Appell, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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comprehend innumerous treatments which practical use is questionable due to the high costs associated, limited efficacy and possible formation of toxic compounds. The use of adsorptive materials has been studied and applied in animal feeds for decades as a method to bind mycotoxins thus making them unavailable for absorption by the animal. As reviewed by Huwig and team (3) the efficacy of these adsorbents against fusariotoxins such as zearalenone, fumonisins and trichothecenes is limited or near zero. This has also been confirmed by in vivo studies (12, 13).An efficient way of detoxification of mycotoxins that can poorly be bound is microbial or enzymatic detoxification (14). In this chapter, the latest advances regarding these biological methods for detoxification of mycotoxins will be thoroughly discussed.

Biotransformation and biodegradation of mycotoxins Biotransformation stands for the conversion of mycotoxins into less toxic molecules by enzymes or microorganisms. As in the case of physical adsorption, this degradation takes place in the gastro-intestinal tract of the animal consuming mycotoxin contaminated feed. Initial research in this field remounts to 40 years ago, when Ciegler et al (15) isolated the first bacteria with aflatoxin-detoxification capability. However, for this mycotoxin, the application of the abovementioned adsorbents was reported to result in almost total protection against aflatoxicosis (3). After that, several research reports about the degradation of other mycotoxins, such as ochratoxins, trichothecenes, zearalenone and fumonisins, were published but for only a few of them the practical use as feed additives has been possible. Trichothecenes biotransformation and non-toxicity of the new metabolite Trichothecenes are a group of more than 200 sesquiterpenes characterized by the 12,13-epoxy-trichothec-9-ene ring system. The epoxy moiety possessed by all trichothecenes has been shown to play a crucial role in their toxicity (14, 16). In comparison to monogastric species, ruminants are generally considered to be less susceptible to the adverse effects caused by contamination of feeds with mycotoxins (17). Based on this knowledge, many detoxification studies on trichothecenes were conducted in the past two decades using rumen fluid (18, 19, 20). First detoxification studies used mixed cultures of anaerobic microorganisms; however, the single strain capable of removing (de-epoxidation the 12,13-epoxy-group to form a double bond was only later on described (21). The gram-positive non-motile, non-spore forming, strictly anaerobic novel bacteria strain from Eubacterium sp. (Eubacterium BBSH 797) was able to transform DON into de-epoxy-DON (DOM-1), a metabolite first described by Yoshizawa et al (22). For the use of BBSH 797 as a feed additive, the fermentation and stabilization processes were optimized with respect to fast growth of the microbe and high biotransformation activity of the resulting



110 product. For enhancement of stability during storage and within the gastrointestinal tract, a three-step encapsulation process was implemented. The toxicity of this new compound was tested using a chicken lymphocyte proliferation assay (LPA) (14). At a DON concentration of 0.15 μg/mL proliferation of lymphocytes was lower in comparison with the control.After adding 0.3 μg/mL to the cells, only one third of them could proliferate whereas at a concentration of 0.63 μg/mL DON the growth of lymphocytes stopped. In the case of DOM-1 only a concentration of 116 μg/mL inhibited proliferation of lymphocyte cells completely. If the concentration values at which the growth of the lymphocytes stopped are compared, it is possible to infer that DOM-1 toxicity is approximately 200 times lower than the parent compound DON. These results are in accordance with previous studies where the biotransformation of DON to de-epoxy-DON had already shown significant loss of cytotoxic activity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) cell-culture test (23). Ochratoxin A biodegradation and non-toxicity of the new metabolite Ochratoxin A consists of a dihydroisocoumarin moiety (the pentaketidederived ochratoxin α) linked through the carboxyl group to phenylalanine (24). After its discovery in 1965 by van der Merwe et al (25) several experiments followed with the aim of finding a suitable microorganism for its detoxification. The Gram-positive soil bacterium Phenylobacterium immobile was shown to degrade ochratoxin A into a less toxic metabolite (26). Later on, Acinetobacter calcoaceticus was found to have the same activity by cleaving the amide bond, thus transforming OTA into ochratoxin alpha (OTα) (27). Other reports show that filamentous fungi from Aspergillus and Rhizopus genera are able to degrade OTA. While A. niger could degrade more than 90% of OTA after a 6-day incubation, the Rhizopus isolates could degrade about 90% of OTA in about 12 days (28). Despite these scientific findings, a suitable mycotoxin-degrading microorganism with practical application in mycotoxin-contaminated feeds was only later on discovered. Following a screening of more than 20 OTA-cleaving microorganisms, the yeast strain associated with the hindgut of lower termites, Trichosporon mycotoxinivorans MTV was isolated and described by Schatzmayr et al (29, 30). OTA was incubated with several OTA-degrading isolates (31)and their degradation rate was compared with that of Phenylobacterium immobile (26). The fastest degraders belonged to the genera of Stenotrophomonas and Trichosporon. Anaerobic isolates were slower in OTA deactivation (complete degradation was detected after 20 hours of incubation). In this experiment Phenylobacterium immobile was the “slowest” strain in terms of OTA-cleavage (31). After being stabilized and applied as lyophilized powder in the feed, the mycotoxin-degrading strain has to regain activity in the gastrointestinal (GI) tract very rapidly, since time available for detoxification is very limited. Therefore, growth and mycotoxin-degradation activity of Trichosporon mycotoxinivorans MTV was optimized. Lyophilized cells grown in an optimized culture medium were able to degrade OTA (200 μg/L) completely into OTα within 1 hour of incubation, whereas cells grown in a standard yeast medium


111 needed more than 5 hours. Studies were developed concerning the toxicity on the degradation metabolite, OTα (14). Growth of macrophages was depressed from 0.741 to 2.222 μg/mL of OTA. At concentrations above 6.667 μg/mL of OTA their growth was completely inhibited. Contrarily, concentrations up to 20 μg/mL of OTα did not affect macrophages growth (14). These results were in accordance with those of other scientific studies were OTα was shown to be non-toxic or at least 500 times less toxic than OTA (32, 33).


Zearalenone biodegradation and non-toxicity of the new metabolite Toxicity of zearalenone relies on the molecule’s resemblance with the sexual female hormone oestradiol, which enables it to couple with the oestrogenic receptors acting as an oestrogen agonist in the brain resulting in severe effects on the reproductive system (34). The history of zearalenone detoxification attempts began in 1988 when the fungus Clonostachys rosea (also known as Gliocladium roseum) was reported to open its lactone ring thus changing its structure and removing its ability to bind to the oestrogenic-binding sites. Two microorganisms, Rhodococcus erythropolis and Norcardia globulera were later on patented as ZON-degrading strains (35). A practical application of these strains has never been reported. Following the studies made with OTA, the yeast strain Trichosporon mycotoxinivorans MTV was incubated with ZON and its activity screened.This led to the reduction of the toxic metabolite into carbon dioxide or into a non toxic metabolite (14). α- and β-zearalenol, which are more estrogenic, could not be detected at the end of the degradation study. The non-toxicity of the newly formed compound was later on tested by an E-screen assay, commonly used system for evaluating the ability of chemicals to induce a hormonal response, based on the ability of MCF-7 cells to proliferate in the presence of estrogens. The cell proliferation of the human estrogen-receptor-positive breast cancer cell line, MCF-7 was quantified by using the colorimetric MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. As this cell line is estrogen-dependent, by incubating it with ZON and separately with the product of ZON incubation with Trichosporon mycotoxinivorans MTV it was possible to compare the growth activity of both inoculates in relation to control. Following the incubation with the test compounds the medium was discarded and 0.6 mg/ml MTT solution was added to the wells. After 4 h incubation the MTT solution was discarded and the purple insoluble formazan solubilized by adding 100 µl/well of lysis buffer (0.5% sodium dodecyle sulphate, 36 mM HCl, and isopropanol acid). After mixing the optical density (OD) was measured at 594 nm using a micro plate reader (BIORAD Model 3550) equipped with a spectrophotometer (BioRad, Veenendaal, the Netherlands). Cell proliferation rate was expressed as (A595 treated cells/A595 of appropriate control) x 100.The standard curve for zearalenone (continuous line in Figure 1) in the E-screen assay indicates a log-normal cell proliferation over the concentration range from 10-6 to 10-11 Mol/L. At higher concentrations ZON becomes toxic to the cells; therefore this concentration range was adopted for the studies of the test compounds. The sample resulting from the ZON incubation with Trichosporon


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mycotoxinivorans MTV (dashed line in Figure 1) did lack the ability to induce any response indicating an estrogenic activity (remaining at baseline values), thus confirming the stable degradation of zearalenone into a non-estrogenic metabolite.

Figure 1. Results of the E-screen assay. The continuous line represents the standard proliferation curve for the incubation of zearalenone with the MCR-7 cell line. The dashed line represents the proliferation curve for the incubation of the MCR-7 cell line with the product of zearalenone and T. mycotoxinivorans incubation.

Fumonisins biodegradation Fumonisins (FUM) are a group of Fusarium toxins which were isolated for the first time in 1988, from moldy maize samples originated from an area in Africa with a high incidence of esophageal cancer (36). The structural similarity of fumonisins to the sphingoid bases sphinganine and sphingosine is critical to their ability to disrupt sphingolipid metabolism (37). Sphingolipids are basically important for the membrane and lipoprotein structure and also for cell regulation and communication (second messenger for growth factors). They are found in great amounts in the brain and in nervous tissue. As in the case of other mycotoxins, also the biotransformation of fumonisins has been an object of scientific research. Two species of “black yeast” found widely in plant debris, Exophiala spinifera and Rhinocladiella atrovirens and a gram negative bacterium from stalk tissue were reported capable of metabolizing fumonisins (38). However, these microorganisms cannot be used as feed additives since they can only perform successfully if fumonisins are the only carbon source present in the media. In addition, E. spinifera is pathogenic and a known causal



113 agent of mycoses. Microorganisms have been isolated from pig intestines, bovine rumen, soil and contaminated maize and screened for FUM-degrading capacity (39). Degradation of the mycotoxin was detected in soil and maize samples. The most promising strains were taxonomically characterized and further tests have been performed to screen the degradation ability in different pH-values, oxygen, temperature, toxin concentration and complex media. One strain was able to completely transform FUM at three different pH-values (5, 7 and 9). All isolates degraded FUM under aerobic conditions. When oxygen was limited, only 2 strains (#144 and 5bfII(2)b) completely degraded FUM in the liquid medium. One strain was capable of completely metabolizing the mycotoxin up to a concentration level of 100mg/L. Degradation experiments in complex media, such as artificially contaminated wheat, maize and beer, revealed two strains (# 144 and 5bfII(2)b) which were capable of completely degrading 2 mg/L FUM (Table 1). Table 1. FUM degradation ability of FUM-degrading isolates in complex media Strain Negative control # 135 # 144 # 151 # 152 5bfII(2)b T2

wheat 0 0 100 5 0 100 100

FUM degradation (%) maize 0 0 100 0 0 100 100

beer 22 0 100 0 2 100 0

SOURCE: Reproduced from Reference 40. Copyright 2006 Wageningen Academic Publishers. Reproduced with permission.

Confirming in vitro findings with in vivo experiments Although in vitro studies represent the basis of research and product development, successful in vivo experiments are crucial to support the effectiveness of any creation on the field. Unfortunately, trials with mycotoxins are very difficult to run and often in controlled experiments, animals show high tolerance to contaminated feeds (17). Nonetheless, the effectiveness of the abovementioned bacteria and yeast strains has been shown in the field.Highly significant (P

Microorganisms and their enzymes for detoxifying mycotoxins posing

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