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Food Control 33 (2013) 352e358

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Food Control

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Degradation of the minor Fusarium mycotoxin beauvericin byintracellular enzymes of Saccharomyces cerevisiae

G. Meca a,*, A. Ritieni b, T. Zhou c, X.Z. Li c, J. Mañes a

a Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, SpainbDepartment of Food Science, University of Naples “Federico II”, via Universitá 100, 80055 Portici, Napoli, ItalycGuelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Rd. W, Guelph, ON N1G 5C9, Canada

a r t i c l e i n f o

Article history:Received 16 January 2013Accepted 23 March 2013

Keywords:BeauvericinIntracellular raw enzymesMycotoxin reductionLCeDADLCeMS/MS

* Corresponding author. Tel.: þ34963544959; fax:E-mail addresses: giuseppe.meca@uv.es, g.meca@v

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.03.035

a b s t r a c t

Beauvericin (BEA) is a cyclic depsipeptide with antibiotic and insecticidal effects. It was discovered forthe first time from the fungus Beauveria bassiana, but more significantly, is produced by several Fusariumstrains, and considered a contaminant of several cereals like corn, wheat and barley.

This study investigated the degradation of BEA by intracellular raw enzymes of four strains ofSaccharomyces cerevisiae, named LO9, YE5, A34, and A17. The BEA at 25 mg/kg in a model solution and incorn flour was co-incubated with the raw enzymes from the four yeast strains, respectively. Thereduction of BEA was evaluated using liquid chromatography coupled to a diode array detector (LCeDAD); the products formed during the co-incubation were determined by liquid chromatographycoupled to mass spectrometry-linear ion trap (LCeMS-LIT). In model solution BEA reduction ranged from83 to 100%. In corn flour treated with the intracellular raw enzymes, the BEA degradation was from 66 to91%. A product resulted from the BEA degradation was identified.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Beauvericin (BEA) (Fig. 1) is a depsipeptide with antibiotic andinsecticidal effects belonging to the enniatin family. It was initiallyisolated from the fungus Beauveria bassiana, but it is also producedby other fungi, including several Fusarium species (Logrieco et al.,1998); it may therefore occur in grains (such as corn, wheat, andbarley) contaminated with these fungi (Jestoi et al., 2004; Logriecoet al., 1998). BEA is inhibitive against gram positive bacteria andmycobacteria, and it is capable of inducing programmed cell deathin mammals as well (Meca et al., 2010). Chemically, BEA is a cyclichexadepsipeptide with alternating methyl-phenylalanyl andhydroxy-iso-valeryl residues. Its ion-complexing capability allowsBEA to transport alkaline earth metal and alkali metal ions acrosscell membranes (Ruiz, Franzova, Juan-Garcia, & Font, 2011).

The potential mycotoxic role of BEA is exemplified by results fromin vitro studies using cell lines. For instance, BEA induces significantcell deaths in insect, murine, and human tumor cell lines (Calo et al.,2003; Mazziotti & Perlmutter, 1998). Furthermore, BEA is a potentand specific inhibitor of cholesterol acyltransferase in rat liver mi-crosomes (Tomoda et al., 1992). In mammalian cell lines, cell deaths

þ3496354954.irgilio.it (G. Meca).

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caused by BEA have been suggested to involve a Ca2þ dependentpathway, in which BEA induces a significant increase in intracellularCa2þ concentration that leads cell death as a result of a combinationof both apoptosis and necrosis (Jow, Chou, Chen, & Tsai, 2004; Linet al., 2005; Logrieco et al., 1998; Nilanonta, Isaka, Kittakoop, &Trakulnaleamsai, 2002; Ojcius, Zychlinsky, Zheng, & Young, 1991).

As regards themethodologies employed for the reduction of thiscontaminant in food, there is only a US patent (Duvick & Rod, 1998)on the biological detoxification of the minor Fusarium mycotoxinBEA in the scientific literature. The inventors achieved 50% BEAreduction by employing a strain of Norocardia glubera as detoxifi-cation agent to wheat kernels with an initial contamination of themycotoxin at 1000 mg/L.

The degradation of Fusariummycotoxins using enzymes purifiedfrom different microbial cultures was evaluated by several authorsin recent years.

For example, Takahashi-Ando, Kimura, Kakeya, Osada, andYamaguchi (2002) studied the enzymatic degradation of Fusariummycotoxin zearalenone (ZEN). The authors evaluated the detoxifi-cation of this bioactive compound with an alkaline hydrolase pro-duced by Clonostachys rosea IFO 7063, evidencing the completetransformation of the mycotoxin in a model solution a 37 �Cincubation.

The detoxification of several Fusarium mycotoxins by a UDP-glucosyltransferase isolated from the small flowering plant

Fig. 1. Chemical structure of BEA.

G. Meca et al. / Food Control 33 (2013) 352e358 353

Arabidopsis thaliana was studied by Peppenberger et al. (2003). Itwas demonstrated that the enzymes, purified from the plant usingchromatographic techniques, detoxified completely mycotoxinsdeoxinivalenol (DON) and 15-acetyl-deoxynivalenol (15-Ac-DON).

The degradation of fumonisisn B1 (FB1) by microbial enzymeswas studied by Heinl et al. (2010). The authors using chromato-graphic techniques isolated two enzymes from a liquid culture ofthe bacterium Sphingopyxis sp. MTA144, capable of detoxifying FB1.

Enzymatic degradations of the FB1 were also studied byHartinger et al. (2011). The authors isolated an enzymatic patternby Sphingopyxis sp. MTA144, with the capacity to degradecompletely the FB1 and related FBs, tested at 10 mM concentrationin model solution.

The degradation of the Fusariummycotoxin ZEN by extracellularenzyme from Acinetobacter sp. SM04 into smaller estrogenicproducts was evaluated by Yu et al. (2011). In particular, using asephadex G-100 column, an active fraction capable to degradein vitro completely 20 mg/mL of ZEN during 6 h incubation wasisolated from the filtrates of the liquid cultures of the bacterialstrain.

The aims of this study were a) to evaluate the BEA degradationby intracellular enzymes of Saccharomyces cerevisiae in a modelsolution and in a food system, and b) to determine the degradationproducts formed through mass spectrometry techniques.

2. Materials and methods

2.1. Chemical and reagents

Acetonitrile and methanol for LC grade were purchased fromFisher Scientific (New Hampshire, USA). Deionized water was ob-tained from aMilli-Q water purification system (Millipore, Bedford,MA, USA). Chromatographic solvents and water were degassed for20 min using a Branson 5200 (Branson Ultrasonic Corp., CT, USA)ultrasonic bath. Casein, trichloroacetic acid (TCA), TriseHCl buffer(pH 7.4), phenylmethanesulfonylfluoride (PMSF), and phosphatebuffer saline (PBS) (pH 7.5) were provided by SigmaeAldrich (St.Louis, USA)

The stock standard solution of BEAwas purchased from SigmaeAldrich (St. Louis, USA). The stock solution was prepared by dis-solving 1 mg of the mycotoxin in 1 mL of pure methanol, obtaininga 1 mg/mL solution. The stock solution was then diluted with puremethanol in order to obtain the appropriated work solutions. Allsolutions were stored in darkness at 4 �C until the LC analysis.

2.2. Strains and methodology

Four strains of S. cerevisiae named LO9, YE5, A34, and A17 wereobtained from the personal collection of Dr. Ting Zhou, the GuelphFood Research Centre (Guelph, Ontario, Canada) and stored in

sterile 18% glycerol at�80 �C. When needed, recovery of the strainswas undertaken by two consecutive subcultures in appropriatemedia prior to use.

The yeasts were cultured in 50 mL sterile plastic centrifugetubes with 20 mL of Potato Dextrose Broth (PDB). The tubes wereincubated at 25 �C in aerobic conditions for 48 h.

2.3. Intracellular protease activity

Intracellular protease activity was measured by the casein assay(Ginther, 1979) with some modification. First, 10 mL of the liquidyeast cultures was centrifuged at 4000� g for 1 min at room tem-perature. The pellet was washed several times with double distilledwater and the yeast cells were disintegrated by sonication. Aftercentrifugation at 30,000� g for 30min, the supernatant was filteredon a Millipore membrane filter (0.45 mm). Next, 1 mL of the su-pernatant was pipetted into a capped Erlenmeyer flask, and then3 mL of casein solution (casein, 0.6%; pH, 7.0) was added. Themixture was then kept at 30 �C for 10 min, and the reaction wasstopped by addition of 3.2 mL of trichloroacetic acid (TCA) solution(TCA, 0.11 M; sodium acetate, 0.22 M; acetic acid, 0.33 M). Themixture was centrifuged at 800� g for 15min at room temperature.The supernatant was filtered on a Millipore membrane filter(0.45 mm) and analyzed by optical density at 280 nm using a UV-752 spectrophotometer. One unit of protease activity was definedas a change of 1.0 in the absorbance at 280 nm/min. The proteinconcentrationwas determined according to themethod of Bradford(Bradford, 1976).

2.4. Intracellular protein extraction

Yeast cells for protein extraction were harvested and washedwith PBS by brief centrifugation (1 min at 4000� g and 4 �C), andimmediately frozen in liquid nitrogen. Typically, cells from 20 mLculture aliquots were collected. Frozen cells were thawed on ice inthree-fold volume of 40 mM TriseHCl buffer (pH 7.4) containing0.5 mM PMSF or a protease inhibitor cocktail, Complete Mini(Roche Diagnostics GmbH, Germany), and disrupted by sonication(Soniprep 150; 4� 30 s bursts at amplitude 7.5mm). Fourmillilitersof the supernatant was directly ultra-centrifuged through a filter of5000 Da (Millipore, Billerica, USA). The dried pellets were storedat �80 �C until use.

2.5. Enzyme activity in model solution

To determine the enzyme activity, BEA (25 mg/mL) was incu-bated in 1 mL of 100 mM phosphate buffer (pH 7.5) with 10 mg ofthe enzymatic extracts corresponding to each strain tested, at 37 �Cfor 4, 9 and 24 h, respectively. At the end of the incubation, thereaction solutions were centrifuged at 4000 rpm, 4 �C, for 5 min.The supernatants were filtered using 0.22 mm filters (Phenomenex,CA) and directly injected in the LCeDAD and LCeMS (Abrunhosa &Venancio, 2007).

2.6. Enzyme activity in the system composed by corn flourcontaminated with BEA

Two grams of corn flour without detectable Fusarium myco-toxins were introduced in 14 mL plastic tubes, and contaminatedwith BEA to a final concentration at 25mg/kg. Twomilliliters of PBScontaining 10mg of the intracellular raw enzymes were introduced(The raw enzymes from each strain were tested individually). Thetubes were incubated at 37 �C for 4, 9 and 24 h.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

LO9 YE5 A34 A17

In

tracellu

lar p

ro

tease activity U

/m

L

Strains

Fig. 2. Protease activity expressed by the intracellular raw enzymes of Saccharomycesstrains.

G. Meca et al. / Food Control 33 (2013) 352e358354

2.7. BEA extraction from corn flour

BEA was extracted according to the procedures described byJestoi (2008). Briefly, 2 g of corn flour samples were extracted with20 mL methanol using an Ultra Ika T18 basic Ultraturrax (Staufen,Germany) for 3 min. The mixture was centrifuged at 4500 g for5 min and then the supernatant was evaporated to dryness with aBüchi Rotavapor R-200 (Postfach, Switzerland). The residue was re-dissolved in 2 mL of extraction solvent. The extract was cleaned upusing a Strata C18-E cartridge (6 mL, 1 g). The cartridge was firstactivatedwith 2� 2mL ofmethanol and conditionedwith 2� 2mLof water before the extract was loaded. The cartridge was thenwashed with 2 � 2 mL of water at a flow rate of 0.5 mL/min till allwater was out. BEA was eluted using 1 mL of methanol.

2.8. LCeDAD analysis

LC analyses of BEA were performed using an Agilent 1100 LCsystem equipped with quaternary pump and a diode array detector(DAD) from Agilent (Santa Clara, USA). A Gemini (150 � 4.6 mm,5 mm) Phenomenex (Palo Alto, CA) columnwas used. LC conditionswere set up using a constant flow at 1.0 mL/min and acetonitrile-water (70:30 v/v) as starting eluent system. The starting ratio waskept constant for 5 min and then linearly modified to 90% aceto-nitrile in 10 min. After 1 min at 90% acetonitrile, the mobile phasewas taken back to the starting conditions in 4 min. BEA wasdetected at 205 nm. All samples were filtered through a 0.22 mmsyringe filter Phenomenex prior to injection (20 mL) onto the col-umn. Mycotoxin identification was performed by comparingretention times and UV spectra of purified extracted samples topure standards. Quantification of mycotoxins was carried out bycomparing peak areas of investigated samples to the calibrationcurve of authentic standards.

2.9. Determination of the BEA degradation product with LCeMS

The separation of BEA was achieved by LC Agilent 1100 (AgilentTechnologies, Santa Clara, California) coupled to a mass spec-trometer, Applied Biosystems/MDS SCIEXQ TRAP TM linear ion trapmass spectrometer (Concord, Ontario, Canada). A Gemini(150 � 2.0 mm, 5 mm) Phenomenex (Torrance, California) columnwas used. LC conditions were set up using a constant flowat 0.3mL/min and acetonitrile/water (80:30, v/v with 0.1% of HCOOH) asmobile phases in isocratic conditionwere used. The instrumentwasconfigured in the positive ion electrospray mode using thefollowing parameters: cone voltage 40 V, capillary voltage 3.80 kV,source temperature 350 �C, desolvation temperature 270 �C andcollision gas energy 5 eV. The analysis of the BEA degradationproduct employing the technique of the liquid chromatographycoupled to the ion trap was carried out using the followingprocedure:

1 Characterization of the compound isolated with the modalityof ER scan, using the m/z range from 200 to 900 Da to obtainthe general spectra of the degradation compound.

2 Characterization of the fragments obtained in the ER scan withthe modality EPI scan to obtain a MS2 scan of a fragment of thedegradation product.

3. Results and discussion

3.1. BEA reduction in model solution

All the strains tested showed an intracellular protease activityranged from 0.74 to 1.14 (Fig. 2). The result from the LC analysis

clearly illustrated that BEA (25 mg/L) was degraded by the intra-cellular raw enzymes from the tested yeast strains (Fig. 3).

As shown in Table 1, in the first 4 h, the BEA reduction by theyeast strains was from 83.0 to 98.5%. The reduction continued toincrease, and at 24 h, reached 99.3% by themost effective strain A34and 89.1% by the least effective strain YE5 (Table 1).

The results suggest that the bioactive compound BEA can bedegraded in model solution by certain strains S. cerevisiae in about24 h.

3.2. BEA reduction in wheat flour

The reduction or degradation of BEA as observed in the abovemodel system was confirmed and validated in a real food systemcomposed of wheat flour. As shown in Table 2, after 4 h incubation,the BEA reduction ranged from 68.3 to 81.4%. Similar to that in themodel system, the reduction rate increased with prolonged incu-bation time. After 24 h incubation, the highest BEA reduction of91.0% was achieved by intracellular raw enzymes from the yeaststrain LO9. The strain A34, the most effective one in the modelsystem, reduced BEA by 83.3% in the food system. The least effectivestrain in the food system was still strain YE5, only reduced BEA by73.5%. As compared to that in themodel solution, the BEA reductionis lower in the food system. This phenomenon might be related tothe complexity of the food system respect to the model solution, inparticular, the proteases responsible of BEA degradation might alsoreact with other proteins presented in wheat flour, reducing theeffectiveness of the enzymatic complex toward the bioactive pep-tide BEA.

This should be the first discovery that the minor Fusariummycotoxin BEA can be degraded by intracellular raw enzymes fromcertain yeast strains that are commonly employed in food tech-nologies although the enzymatic degradation of other mycotoxinshave been reported by many authors. Takahashi-Ando et al. (2002)studied the degradation of the estrogenic Fusariummycotoxin ZEN,employing enzymatic extracts produced by fermentation of thefungus C. rosea in liquid medium of YG. Each fraction (89 mL) wasmixed with 25 mg of ZEN and 10 mL of 1 M Tris/HCl (pH 9.5). Thetotal volume of the reaction was adjusted to 100 mL with water andthe reaction mixture was incubated at 37 �C. Although the purifiedenzymewas able to detoxify ZEN at pH 7, its maximum activity wasobserved at pH 9e10. Considering that ZEN is stable at pH 9.5, theenzyme responsible for the detoxification appeared to be a hy-drolase (designated as ZHD101) that cleaves the lactone ring enableto degrade totally the mycotoxin tested.

Poppenberger et al. (2003) reported the isolation and character-ization of a gene from A. thaliana encoding a UDP-glycosyltransferase

Fig. 3. LCeDAD chromatograms of a) BEA present in model solution before the treatment with the intracellular row enzymes and b) after the enzymatic treatment with the rowenzymes of Saccharomyces cerevisiae A34.

G. Meca et al. / Food Control 33 (2013) 352e358 355

that is able to detoxify deoxynivalenol. The enzyme, previouslyassigned the identifier UGT73C5, catalyzes the transfer of glucosefrom UDP-glucose to the hydroxyl group at carbon 3 of deoxy-nivalenol. This deoxynivalenol-glucosyltransferase (DOGT1)was alsofound to detoxify the acetylated derivative 15-Ac-DON, whereas noprotective activity was observed against the structurally similarnivalenol (NIV).

Heinl et al. (2010), evaluated that the detoxification of themycotoxin FB1 comprises at least two enzymatic steps, an initialdesterification reaction, followed by deamination of the resultinghydrolyzed fumonisin B1 (HFB1). The authors demonstrated that twogenes of the bacterium Sphingopyxis sp. MTA144 were responsible ofthe production of two enzymes that detoxify completely the FB1. Thedetoxification tests were carried out in a buffer composed by 20mMof tris HCL pH 8.0, 0.1 mg/mL BSA, containing 5 mM of FB1 at 30 �Cincubation. The first gene encodes a protein which shows similarityto carboxylesterases, type B. The second gene encodes a polypeptidehomologous to aminotransferases, class III. The two genes wereisolated and expressed heterologously.

The recombinant carboxylesterase was shown to catalyze thedeesterification of FB1 to hydrolyzed FB1. The heterologouslyexpressed aminotransferase was shown to deaminate hydrolyzedFB1 in the presence of pyruvate and pyridoxal phosphate. The au-thors demonstrated that the consecutive action of these two en-zymes is sufficient for the total FB1 detoxification.

Hartinger et al. (2011) studied the FB1 degradation by amino-transferase FumI isolated through chromatographic techniques by a

Table 1BEA reduction in model solution during the reactionwith the microbial intracellularrow enzymes.

Strains % BEA reduction

4 h 9 h 24 h

S. cerevisiae LO9 85.5 � 1.0 88.5 � 1.3 89.4 � 2.9S. cerevisiae YE5 83.0 � 1.5 85.4 � 1.2 89.1 � 1.5S. cerevisiae A34 98.5 � 2.0 98.3 � 2.1 99.3 � 1.6S. cerevisiae A17 93.7 � 2.6 96.2 � 2.3 97.0 � 2.0

liquid culture fermented by Sphingopyxis sp. MTA144. The authorsobtained the completely degradation of the mycotoxin assayedcarry out the test in a model solution composed by 20 mM of TriseHCL pH 7.4, 50 mM NaCl, 2 mM CaCl2, 20 mM pyridoxal phosphate(PLP), and 0.1 mg/mL bovine serum albumin (BSA) spiked with 10and 15 mM of FB1 and HFB1 during 3 h incubation with the purifiedenzyme. The authors discovered that FB1 is substrate of thefumonisin arboxylesterase FumD, which catalyzes hydrolyticcleavage of both tricarballylic acid (TCA) chains off the core chain toproduce HFB1 and tricarballylic acid. Aminotransferase FumItransfers the 2-amino group from HFB1 to pyruvate, producing 2-keto-HFB1 and alanine.

Microorganisms and enzymes responsible for detoxification oftrichothecenes by deepoxidation, hydroformylation, hydroxylation,hydrolysis and glucuronidation may be ubiquitous in nature (He,Zhou, Young, Boland, & Scott, 2010). Application of microbialdetoxification agents (living cells) in the human food and animalfeed industries seems to be diminishing because of regulatory,toxicological, and consumer considerations. Thus, applications ofdetoxification enzymes and genes can represent alternate detoxi-fication methods. Purification and characterization of detoxifica-tion enzymes from various sources (microorganisms, plants andmammalian tissues) can lead to better understanding of these en-zymes, and their properties and kinetics. Advanced molecularbiology and genetic engineering techniques can be used to identifyand characterize genes involved in these detoxification enzymes(Altalhi, 2007; Altalhi & El-Deeb, 2009; Takahashi-Ando et al.,

Table 2BEA reduction in in corn flour during the reaction with the microbial intracellularrow enzymes.

Strains % BEA reduction

4 h 9 h 24 h

S. cerevisiae LO9 80.9 � 2.5 86.3 � 1.3 91.0 � 1.0S. cerevisiae YE5 68.3 � 1.6 71.1 � 1.0 73.5 � 2.3S. cerevisiae A34 81.4 � 1.4 81.7 � 0.8 83.0 � 2.2S. cerevisiae A17 76.2 � 1.9 82.1 � 2.1 86.4 � 1.4

Fig. 4. a) LCeMS-LIT chromatogram of the BEA enzymatic degradation product present in the model system treated with the intracellular enzymes of Saccharomyces cerevisiae A34and b) ER spectrum of the enzymatic degradation product.

G. Meca et al. / Food Control 33 (2013) 352e358356

Fig. 5. EPI profile of the ion corresponding at m/z 573.3 (MS2) evidenced in the ER spectrum.

G. Meca et al. / Food Control 33 (2013) 352e358 357

2004). Once the genes are obtained, they may be cloned andexpressed in crops to develop varieties resistant to mycotoxinproduction or which detoxify mycotoxins, in order to preventtrichothecenes from entering human and animal food chains. Thedetoxification genes may be cloned and expressed in microorgan-isms to produce recombinant microorganisms that are suitable inan industrial scale enzyme production and purification (Altalhi &El-Deeb, 2009). These detoxification enzymes should have greatpotentials to eliminate trichothecenes in the human and animalfood chains.

3.3. Potential mechanism of BEA degradation

The formation of new products from BEA degradation by mi-crobial intracellular proteases was studied using the techniques ofthe LCeMS-LIT, which made possible to study the structure of anewly formed compounds in high precision level. Fig. 4a evidencesthe LCeMS-LIT chromatogram of the BEA degradation accom-plished by the intracellular enzymes of S. cerevisiae A34. In thechromatogram, in addition to the BEA peak with a retention time(RT) of 24.65, another peak appears at the RT of 14.89, indicatingthe presence of a BEA degradation product.

Fig. 4b shows the ER spectrum of this product, where are pre-sent several diagnostic fragments that confirm the degradation ofBEA. In particular the signal at m/z 573.3 correspond to the BEAwith the loss of N-Phe, that is an important structural amino acid ofthis compound more two molecules of water. The formation of thisnew product is confirmed by other fragments like the m/z 515.4that represents the m/z 573.3 with the loss of an isopropyl group(-i-Pr)þ, and by the fragment with a m/z of 471.4 that corresponds

to the ion with m/z 573.3 with the loss of another structural com-pound of BEA that is the hydroxyvaleric acid (HyLv). Anotherimportant fragment is the signal withm/z 429.4 that represents theion with m/z 573.3 with the loss of a structural compound of BEArepresented by a N-Phe. To confirm that the ion with m/z 573.3 isoriginated by BEA, this fragment was studied with the technique ofEPI (m/z 50e550) (Fig. 5) that permits to know the MS2 of 573.3. InFig. 5 is possible to evidence in the right part of the spectrum thesame ions evidenced in the ER spectra that correspond to thestructural components of the ion with a m/z 573.3, and also twodiagnostic signals with a m/z of 114.1 and 154.1. These two frag-ments evidence the loss of HyLv and (HyLv þ 2H2O)þ by thecompound with a m/z of 573.3. Considering that the HyLv is astructural compound of the bioactive compound BEA and is presentin the MS and MS2 spectra of a degradation product, these dataconfirm that the origin of the ion 573.3 is related by a biologicaldegradation of the mycotoxin BEA.

The degradation product was detected in both the model solu-tion and the food system.

BEA is a mycotoxin of relatively minor importance in terms of itsoccurrence and toxicity; therefore methods for detoxifying orreducing BEA have not been developed. The present study presentsa novel approach in reducing BEA contamination in foods and feedusing enzymatic complexes by yeast strains used commonly in foodproductions. This is the first report where a biological degradationof BEA by intracellular raw enzymes produced by several Saccha-romyces strains was studied. Further investigation will be focusedon the characterization, using proteomic techniques, of the en-zymes responsible of the BEA degradation and also on the toxicityof the degradation product employing cell models.

G. Meca et al. / Food Control 33 (2013) 352e358358

Acknowledgments

This research was supported by the Ministry of Science andInnovation (AGL2010-17024), and by the pre PhD program of Uni-versity of Valencia “Cinc Segles”. The microbial degradationresearch was conducted in Dr. Ting Zhou’s laboratory at GuelphFood Research Center, Agriculture and Agri-Food Canada.

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