Pawel Pohl

Determination and fractionation of metals in beer -a review

Keywords: Metals analysis -AAS, Metals analysis -ICP, Metals analysis -ICP/MS Beer, metals, determination, fractionation, atomic spectrometry

Major, minor and trace metals are important in beer fermentation since they supply the appropriate environment for yeast growth and influence yeast metabolism. A real concern is the content of Cu and Fe, which are involved in beer conditioning and ageing through reactions resulting in formation of reactive oxygen species. The reactive oxygen species readily oxidize organic compounds present in beer, changing the quality of foaming and the flavor stability of beer. In view of brewing technology and beer processing, knowledge regarding functions of metals and their speciation in brewing liquors and beer is of special significance. Metals in beer also have a certain nutritional importance but their actual effect related to beer consumption depends on the type of species they form with low and high molecular mass organic ligands which naturally occur in beer. This review covers the determination and fractionation of metals in beer using atomic spectrometry methods. Special attention is drawn to the role of metals in beer and brewing, possible metal associations, methods of beer preparation before analysis on the total metal content and approaches to metal partitioning in beer.

Introduction

Sources of metals in beer

Beer is a product of a yeast alcoholic fermentation of extracts of malted cereals, usually barley malts, with or without a starchy material, and to which hops are added. All natural components used for brewing, including water, cereals, barleys and yeasts, are the main endogenous sources of metals in beer. For that reason, the mineral composition of beer reflects the composition of ingredients used for brewing and refers to processes involved in a beer production. The content of metals is variable and depends on quality of substrates taken, type of beer brewed and country of origin of beer [START_REF] Hardwick | The Properties of Beer[END_REF][START_REF] Moll | Water[END_REF][START_REF] Goldammer | The Brewer's Handbook[END_REF][START_REF] Baxter | Beer: Quality, Safety and Nutritional Aspects[END_REF][START_REF] Briggs | Brewing Science and Practice[END_REF]).

Metals in beer may also come from other substances added during brewing so as to control fermentation and maturation processes. Another exogenous source of metals in beer can be a contamination from brewery equipments, i.e. pipes, fluid lines, vessels and tanks, used for beer handling, including fermentation, conditioning, filtration, carbonating and packing. Also containers (kegs, casks, cans), in which a final quality product is stored and transported, can be a potential source of beer contamination. Indeed, moderately acidic brewing liquors and beer (pH about 4.2 on average) can to a large extent contribute to a metal ions ingestion, especially in case of Al cans, kegs and casks [START_REF] Sharpe | Content, chemical speciation, and significance of aluminum in beer[END_REF][START_REF] Vela | Detection of aluminum residue in fresh and stored canned beer[END_REF][START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF].

Aluminum can liners deterioration and an interaction of beer with containers usually leads to an increase in concentrations of Al, Co, Cr, Cu, Fe or Ni, which readily migrate to the beer. In the case of Al [START_REF] Vela | Detection of aluminum residue in fresh and stored canned beer[END_REF], it can be presumed that the longer the length of beer storage, the higher concentrations of metals that are found in the beer.

Additionally, the higher the temperature of storage, the more rapid is the rate of can 3 corrosion which takes place, regardless of its coating, and the higher the accumulation of metals in beer that occurs.

Metals in brewing

Besides the nutritional value of metals from beer with respect to their potential relationship and contribution to daily diary nutrition requirements, they are also very important and influential in the brewing process itself. When considering the growth and metabolism of yeasts, many metals are essential co-factors for numerous fermentative enzymes, being necessary components of transport systems fulfilling different charge-balancing or structural roles. Concentrations of Ca(II), Co(II), Cu(II), Fe(III), K(I), Mo(II), Mn(II), Mg(II), Ni(II) and Zn(II) ions required for the appropriate yeasts growth are reasonably small. At high concentrations, these metals are usually toxic or exert a salt stress on the yeasts as in case of Na(I) for example [START_REF] Briggs | Brewing Science and Practice[END_REF].

With respect to improvements in yield and a fermentative capacity, as well as a maintaining consistency of a product quality, metal metabolism of yeasts is of special interest to brewers [START_REF] Walker | Magnesium, calcium, and fermentative metabolism in industrial yeasts[END_REF][START_REF] Chandrasena | Use of response surfaces to investigate metal ion interactions in yeast fermentations[END_REF]. Normally, the metal ion composition of brewing liquors notably changes during brewing and the rate of this alteration depends on raw materials used for beer production and conditions of the brewing process. The required metal uptake by yeast cells normally relies on the concentration of particular ions in the growth media and, to a great degree, on their bioavailability. The latter factor depends on both solubility and properties of different ion-complexing ligands originated from raw materials. Free metal ions are the most bioavailable to yeast cells, but chelating and adsorbing constituents present in wort and Mg(II) or Zn(II) ions has a detrimental effect on a yeast growth kinetics and a fermentation performance [START_REF] Walker | Magnesium, calcium, and fermentative metabolism in industrial yeasts[END_REF][START_REF] Chandrasena | Use of response surfaces to investigate metal ion interactions in yeast fermentations[END_REF]. Information on the role of some specific major and minor metals in yeast metabolism and the fermentative and sensory performance of beer is given in Table I [START_REF] Hardwick | The Properties of Beer[END_REF][START_REF] Moll | Water[END_REF][START_REF] Walker | Magnesium, calcium, and fermentative metabolism in industrial yeasts[END_REF][START_REF] Chandrasena | Use of response surfaces to investigate metal ion interactions in yeast fermentations[END_REF][START_REF] Goldammer | The Brewer's Handbook[END_REF][START_REF] Baxter | Beer: Quality, Safety and Nutritional Aspects[END_REF][START_REF] Briggs | Brewing Science and Practice[END_REF]).

Nevertheless, it is evident that with reference to beer stability and quality not only the mineral composition of beer and brewing liquors should be controlled but also the speciation forms of the metals.

Beer ageing and haze formation

It is recognized that in the presence of Cu + and Fe 2+ ions, stable molecules of O 2 dissolved in beer capture electrons and form superoxide anions (O 2 -). Usually, resulting metal cations (Cu 2+ , Fe 3+ ) can be reduced again to respective lower oxidation state forms by such pro-oxidant molecules as some polyphenols [START_REF] Kaneda | Behavior and role of iron ions in beer deterioration[END_REF]Bamforth et al. 1993, Uchida and[START_REF] Uchida | Improvement for oxidative flavor stability of beer -role of OHradical in beer oxidation[END_REF]Andersen and Skibstead 1998;[START_REF] Kaneda | Beer staling mechanism[END_REF][START_REF] Vanderhaegen | The chemistry of beer aging -a critical review[END_REF]. By further protonation, O 2 -anions form perhydroxyl radicals (OOH [START_REF] Kaneda | Beer staling mechanism[END_REF][START_REF] Vanderhaegen | The chemistry of beer aging -a critical review[END_REF].

The reactive oxygen containing species (O 2 -, OOH It is also documented that polyphenolic compounds contribute to beer ageing reactions. Some of polyphenols are pro-oxidants capable of transferring electrons to metal ions and reducing Cu 2+ and Fe 3+ to respective Cu + and Fe 2+ forms, which indirectly stimulates the activation of O 2 in beer. In this process, polyphenols become radicals and can also react with other beer constituents leading to their deterioration or can decompose, producing various off-flavors. On the other hand, polyphenols act as anti-oxidants capturing Cu and Fe ions and forming their stable complexes. Apart from polyphenols, aminoacids, phytic acids and melanoidins in brewing liquors and beer have also this ability and sequestrate metal ions to promote the active oxygen radical species formation [START_REF] Vanderhaegen | The chemistry of beer aging -a critical review[END_REF].

Free metal ions are supposed to affect formation of a protein-polyphenol haze (chill haze) resulting from a cross-linkage of proteins by low molecular weight polyphenols [START_REF] Siebert | Formation of protein-polyphenol haze in beverages[END_REF][START_REF] Mcmorrough | Haze formation and shelf-life prediction for lager beer. Food Technology 53:58-62. accumulation and release of metals causing beer instability[END_REF] when polyphenols subsequently interact with proteins after their polymerization, a "permanent haze" of particle size between 1 and 10 µm is formed in beer.

Finally, it should be noted that the polyphenol polymerization is strongly promoted by their oxidation [START_REF] Kaneda | Effect of free radicals on haze formation in beer[END_REF][START_REF] Kaneda | Behavior and role of iron ions in beer deterioration[END_REF]Bamforth 1999b).

Indeed, when the rate of the reactive oxygen species formation, in reactions activated and catalyzed by Cu and Fe ions, is high, the haze readily appears because of polyphenols oxidation.

Determination of metals in beer

Total metal content

The major metals of beer are Ca (4-140 mg l -1 ), K (18-1100 mg l -1 ), Mg (24-270 mg l - 1 ) and Na (1-230 mg l -1 ). To the groups of minor and trace metals belong Al (0.005-2.2 mg l -1 ), Ba (0.01-0.07 mg l -1 ), Cd (0.0002-0.7 mg l -1 ), Co (<0.0008 mg l -1 ), Cr (<0.02 mg l -1 ), Cu (0.008-0.8 mg l -1 ), Fe (0.02-1.6 mg l -1 ), Hg (<0.3 mg l -1 ), Mn (0.03-0.8 mg l -1 ), Ni (<0.3 mg l -1 ), Pb (0.0008-0.03 mg l -1 ), Sr (0.1-0.7 mg l -1 ) and Zn (0.001-1.5 mg l -1 ). The concentrations of major as well as some minor and trace metals in beers coming from different countries are given respectively in Table II [START_REF] Bamforth | Nutritional aspects of beer -a review[END_REF]. However, it should be also noted that although the information on the total metal content in beer is advantageous for the estimation of the metal nutrient uptake source, metals bioavailability from beer to humans and their absorbability in the gastric system crucially depends on speciation forms in which they are present in beer [START_REF] Sharpe | Content, chemical speciation, and significance of aluminum in beer[END_REF][START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF][START_REF] Pohl | Fractionation analysis of manganese and zinc in beers by means of two sorbent column system and flame atomic absorption spectrometry[END_REF]. For instance, it is indicated that Al (where excessive intake is acknowledged to be associated with Alzheimer's disease) is predominantly present in beer as citrate and phosphate complexes and that only a small portion of the total Al in beer is transported through the gut wall. Consequently, beer plays rather a minor part in the uptake of Al from the diet [START_REF] Sharpe | Content, chemical speciation, and significance of aluminum in beer[END_REF].

The determination of the total metal composition of beer, including major, minor and trace metals, is of particular interest to brewers and consumers. Depending on the concentration and type, metals may be essential or toxic to the human body and can also affect the brewing process and beer quality in view of flavor stability and haze formation [START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF][START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF][START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF][START_REF] Vinas | Determination of cadmium, aluminium, and copper in beer and products used in its manufacture by electrothermal atomic absorption spectrometry[END_REF][START_REF] Bellido-Milla | Beer digestions for metal determination by atomic spectrometry and residual organic matter[END_REF][START_REF] Pohl | Pre-concentration of Cd, Co, Cu, Ni and Zn using different offline ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection[END_REF][START_REF] Asfaw | Direct analysis of beer by ICP-AES: a very simple method for the determination of Cu, Mn and Fe[END_REF][START_REF] Nascentes | Direct determination of Cu, Mn, Pb, and Zn in beer by thermospray flame furnace atomic absorption spectrometry[END_REF][START_REF] Llobat-Estelles | Detection of bias errors in ETAAS: Determination of copper in beer and wine samples[END_REF][START_REF] Onate-Jaen | Spectrophotometric methods to differentiate beers and evaluate beer ageing[END_REF][START_REF] Pohl | Fractionation analysis of manganese and zinc in beers by means of two sorbent column system and flame atomic absorption spectrometry[END_REF].

Some metals may be harmful above a certain concentration (Al, Cd, Hg, Pb), some of them are regarded to influence the foam and flavor quality (Cu, Fe, Mn), others are essential, and have advantageous effect on human health and well-being (Fe, Zn).

Accordingly, in regard to different health and/or disease implications of metals, their nutritional value and impact on beer quality and sensory properties, the total allowed 2001;[START_REF] Briggs | Brewing Science and Practice[END_REF].

Measurement methods

Due to quantities of metals in beer, typically varying in the range from 10 -3 (Cd, Co, Cu, Cr, Pb) through 10 -2 -10 0 (Al, Ba, Fe, Mn, Sr, Zn) up to 10 1 -10 2 mg l -1 (Ca, K, Mg, Na), methods of choice for their determinations are foremost atomic spectrometric techniques. These include flame atomic absorption spectrometry (F-AAS) [START_REF] Fantozzi | In vitroantioxidant capacity from wort to beer[END_REF][START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Bellido-Milla | Beer digestions for metal determination by atomic spectrometry and residual organic matter[END_REF][START_REF] Onate-Jaen | Spectrophotometric methods to differentiate beers and evaluate beer ageing[END_REF]Pohl andPrusisz 2007, Pohl 2007b), electrothermal atomic absorption spectrometry (ET-AAS) [START_REF] Wagner | Determination of zinc in wort and beer by graphite furnace atomic absorption spectroscopy[END_REF][START_REF] Sharpe | Content, chemical speciation, and significance of aluminum in beer[END_REF][START_REF] Wagner | Determination of lead in beer using zeeman background-corrected graphite furnace atomic absorption spectrometry[END_REF][START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF][START_REF] Vinas | Determination of cadmium, aluminium, and copper in beer and products used in its manufacture by electrothermal atomic absorption spectrometry[END_REF][START_REF] Asfaw | Direct analysis of beer by ICP-AES: a very simple method for the determination of Cu, Mn and Fe[END_REF][START_REF] Llobat-Estelles | Detection of bias errors in ETAAS: Determination of copper in beer and wine samples[END_REF], and inductively coupled plasma optical emission spectrometry (ICP-OES) [START_REF] Matsushige | Determination of trace elements in Brazilian beers by ICP-AES[END_REF][START_REF] Vela | Detection of aluminum residue in fresh and stored canned beer[END_REF][START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF][START_REF] Bellido-Milla | Beer digestions for metal determination by atomic spectrometry and residual organic matter[END_REF][START_REF] Pohl | Pre-concentration of Cd, Co, Cu, Ni and Zn using different offline ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection[END_REF][START_REF] Asfaw | Direct analysis of beer by ICP-AES: a very simple method for the determination of Cu, Mn and Fe[END_REF][START_REF] Sedin | Elemental analysis of beer and wort by inductively coupled plasmaatomic emission spectroscopy[END_REF][START_REF] Sedin | Elemental analysis of beer and wort by inductively coupled plasmaatomic emission spectroscopy[END_REF]).

Other spectrometric techniques proposed for measuring the total metal contents in beer are integrated atom trap flame atomic absorption spectrometry (IAT-F-AAS) (Matusiewicz and Kopras1997), thermospray flame furnace atomic absorption spectrometry (T-FF-AAS) [START_REF] Nascentes | Direct determination of Cu, Mn, Pb, and Zn in beer by thermospray flame furnace atomic absorption spectrometry[END_REF], and inductively coupled plasma mass spectrometry (ICP-MS) [START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF].

Sample treatment

Degassing. As an analytical sample, beer is a very complex matrix with a relatively high content of different organic compounds originated from brewery processing (mostly carbohydrates and proteins) and saturated with CO 2 . To take an exact sample volume for analysis, beer requires degassing at first. The simplest way to degas beer is to leave it loosely capped for at least 24 hours after an initial addition of concentrated HNO 3 [START_REF] Sharpe | Content, chemical speciation, and significance of aluminum in beer[END_REF]. Usually, immersing a respective beer aliquot in an ultrasonic bath for 5 to 20 min performs degassing equally well. Apart from a sonication, beer can be also degassed by exposing to microwaves in a pressurized closed vessel microwave oven, operated at a moderate power for several minutes [START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Bellido-Milla | Beer digestions for metal determination by atomic spectrometry and residual organic matter[END_REF], by a percolation of an inert gas through a sample aliquot for an hour [START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF][START_REF] Asfaw | Direct analysis of beer by ICP-AES: a very simple method for the determination of Cu, Mn and Fe[END_REF] or by passing a beer portion through a 0.45 µm pore size membrane filter or a dance filter paper [START_REF] Wagner | Determination of zinc in wort and beer by graphite furnace atomic absorption spectroscopy[END_REF][START_REF] Wagner | Determination of lead in beer using zeeman background-corrected graphite furnace atomic absorption spectrometry[END_REF][START_REF] Matusiewicz | Methods for improving the sensitivity in atom trapping flame atomic absorption spectrometry: analytical scheme for the direct determination of trace elements in beer[END_REF]Pohl andPrusisz, 2007, Pohl 2007b).

Wet digestion. The presence of organic constituents, even at low levels resulting from a dilution of beer samples with water, is recognized as a noticeable source of matrix effects in the transport of samples to an atomization cell and the atomization processes the occur therein [START_REF] Nascentes | Direct determination of Cu, Mn, Pb, and Zn in beer by thermospray flame furnace atomic absorption spectrometry[END_REF]. As a result, destruction of the organic matter and matrix dissolution is normally carried out before beer analysis using atomic spectrometry techniques mentioned above. [START_REF] Pohl | Pre-concentration of Cd, Co, Cu, Ni and Zn using different offline ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection[END_REF] or HNO 3 with HCl [START_REF] Vela | Detection of aluminum residue in fresh and stored canned beer[END_REF]) are also used for that purpose. Rather more rarely beer can be ashed in a muffle furnace (at 550 o C) and the resulting residue dissolved afterwards using HCl and water [START_REF] Fantozzi | In vitroantioxidant capacity from wort to beer[END_REF].

Digestion protocols based on use of a closed vessel temperature programmable microwave oven are also often used. Usually, beer is treated with HNO 3 [START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF] or a mixture of HNO 3 with H 2 O 2 , where in the latter case the reagents are added simultaneously [START_REF] Asfaw | Direct analysis of beer by ICP-AES: a very simple method for the determination of Cu, Mn and Fe[END_REF] or sequentially [START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Bellido-Milla | Beer digestions for metal determination by atomic spectrometry and residual organic matter[END_REF][START_REF] Llobat-Estelles | Detection of bias errors in ETAAS: Determination of copper in beer and wine samples[END_REF][START_REF] Onate-Jaen | Spectrophotometric methods to differentiate beers and evaluate beer ageing[END_REF].

Certainly, the use of microwave heating systems has several advantages over the traditional open vessel dissolution approach. It considerably diminishes the time of the digestion protocol and the consumption of reagents, in addition, reduces a risk of an analyte loss and/or a sample contamination [START_REF] Bellido-Milla | Beer digestions for metal determination by atomic spectrometry and residual organic matter[END_REF]). In consequence, better analysis reproducibility is achieved.

An interesting beer digestion procedure has been described by [START_REF] Matusiewicz | Methods for improving the sensitivity in atom trapping flame atomic absorption spectrometry: analytical scheme for the direct determination of trace elements in beer[END_REF], in which beer samples, after addition of HNO 3 and H 2 O 2 , are exposed to UV radiation for 4 hours.

Unfortunately, regardless of the mineralization procedure used, there can be still some remaining organic matter in the samples that may have an effect on the analyte responses, even when microwave assisted digestion procedure is executed. Direct analysis. To simplify and improve speed of beer analysis, digestion can be avoided. In case of measurements with ICP-OES, a direct aspiration of analyzed beer samples is often preferred, however, lower signal-to-background ratios are usually achieved and a high supplied power is required for plasma operation [START_REF] Pohl | Pre-concentration of Cd, Co, Cu, Ni and Zn using different offline ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection[END_REF][START_REF] Asfaw | Direct analysis of beer by ICP-AES: a very simple method for the determination of Cu, Mn and Fe[END_REF][START_REF] Sedin | Elemental analysis of beer and wort by inductively coupled plasmaatomic emission spectroscopy[END_REF][START_REF] Sedin | Elemental analysis of beer and wort by inductively coupled plasmaatomic emission spectroscopy[END_REF]). Undiluted beer samples are also analyzed using ET-AAS [START_REF] Wagner | Determination of zinc in wort and beer by graphite furnace atomic absorption spectroscopy[END_REF][START_REF] Wagner | Determination of lead in beer using zeeman background-corrected graphite furnace atomic absorption spectrometry[END_REF].

More frequently, a reasonably dilution (from 1/1 to 1/10) of beer aliquots with water, especially when using F-AAS or ET-AAS for measurements, is necessary in order to minimize possible physical and/or chemical matrix interferences accompanying Another approach to beer analysis by ET-AAS without need for comparatively long sample dissolution or a respective sample dilution has been reported by [START_REF] Vinas | Determination of cadmium, aluminium, and copper in beer and products used in its manufacture by electrothermal atomic absorption spectrometry[END_REF]. Only oxidizing agents (HNO 3 and H 2 O 2 ) are added to beer samples maintaining a proper oxidizing environment during the atomization and preventing an accumulation of carbonaceous residues inside a graphite tube. This method has been proved to give very repeatable and reliable results.

Differentiation and classification of beer

Information on the total metal content of beer is recognized to be valuable for differentiation and classification of beer [START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF][START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF]. This is because metals are very good descriptors reflecting the composition of natural raw products (water, cereals, hops and yeasts) used for brewing and indicating processes involved in beer manufacturing, as well as beer storage and ageing. Among other metals, Mg, Mn and K are established to be the most important variables for such classification [START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF].

Using PCA it has been indicated that there exists a certain degree of associations between some metals and they tend to group together in beer, i.e. Cs-Hg, Cd-Co, Cr-Cs, Fe-Zn or Mn-V [START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF]). These metal cluster correlations found possibly result from a supposition of two main sources of metal intake in beer, i.e. the endogenous source associated with raw materials used for brewing and the exogenous source originated from contaminations taking place during the production and storage of beer.

Analysis of variance between the groups (ANOVA) is also a valuable statistical method for beer characterization according to type, country of origin and container. It is suitable as well for controlling the quality of final products achieved with respect to the taste and flavor stability. Accordingly, applying ANOVA to 25 different beers, in which Al, Ca, Cu, Fe, Mg, Mn and Zn were determined, it has been found that the [START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF]. In addition, notable differences in content of Ca, Cu, Fe, Mg, Mn and Na are observed among lager, stout, wheat and ale beers, demonstrating that the metal intake is differentiated according to type of beer and distinctive technology of its production.

Fractionation of metals in beer

Metal associations in beer

Although studies devoted to characterization and quality control of beer related to the total mineral composition are obviously valuable, it should be highlighted that the effect of metals on beer stability and wholesomeness does not depend on their concentrations but foremost on type of metal associations preset in beer. Different low and high molecular mass organic compounds in beer can bind metals, while an existing equilibrium between complexed and non-complexed metal ions is responsible for beer quality and nutritional properties.

Indeed, beer contains various classes of natural compounds, including polyphenolics, proteins, amino acids or other organic species that have a high capacity of binding the metals through donor N, O and S atoms. Thus, metals in beer can be present as non-complexed cations and in the form of complexes of different stability, and therefore, of different bioavailability and toxicity to humans [START_REF] Montanari | Organic and phenolic acids in beer[END_REF][START_REF] Gorinstein | Comparative contents of some phenolics in beer, red and white wines[END_REF][START_REF] Cortacero-Ramirez | Analysis of beer components by capillary electrophoretic methods[END_REF][START_REF] Nardini | Determination of free and bound phenolic acids in beer[END_REF][START_REF] Khatib | Application of two-dimensional J-resolved nuclear magnetic resonance spectroscopy to differentiation of beer[END_REF]).

Due to a crucial role of Cu(I) and Fe(II) ions in the process of molecular O 2 activation and further oxidation of organic compounds, responsible for beer stability technology and beer processing [START_REF] Irwin | The role of copper, oxygen, and polyphenols in beer flavor instability[END_REF]Mochaba et al. 1996;[START_REF] Andersen | Electron spin resonance spin trapping identification of radicals formed during aerobic forced aging of beer[END_REF][START_REF] Montanari | Organic and phenolic acids in beer[END_REF][START_REF] Blanco | Chelation of aqueous iron(III) by 2-acetyl-1,3-cyclohexanedione and beer ageing[END_REF][START_REF] Morales | Iron-binding ability of melanoidins from food and model systems[END_REF]. During the storage of beer, Cu and Fe can form very stable complexes with amino acids, polyphenols, and melanoids, being the products of Maillard reactions. This capture of Cu and Fe ions and association with organic compounds certainly decrease the rate of formation and activity of reactive oxygen species (Bamforth 1999a;[START_REF] Blanco | Chelation of aqueous iron(III) by 2-acetyl-1,3-cyclohexanedione and beer ageing[END_REF][START_REF] Vanderhaegen | The chemistry of beer aging -a critical review[END_REF]). Non-complexed Cu and Fe ions may have rather a negative effect on beer quality, contributing to changes in beer stability and chemical deterioration with ageing. On account of this special role of Cu and Fe, the content of these metals determines the type of beer ageing characteristics and final beer aroma and taste [START_REF] Vanderhaegen | The chemistry of beer aging -a critical review[END_REF]).

Speciation of Mn or Zn has been less studied. The ability of Mn(II) ions to form complexes with organic compounds is considered to be rather low (Onate-Jaen et al.

2006

). Although divalent Mn cations have not been reported to participate in radical generation and oxidative staling reactions, chemical properties of this metal and association with high molecular polyphenolic and flavonoid constituents tend to a conclusion that Mn effect on beer colloidal and flavor instability is possible [START_REF] Pohl | Fractionation analysis of manganese and zinc in beers by means of two sorbent column system and flame atomic absorption spectrometry[END_REF]Pohl 2007b). Non-complexed Mn ions may enhance the action of Cu and Fe in catalyzing staling reactions in beer, however, a relatively low concentration of Mn as compared to Cu and Fe may mask this effect (Mochaba et al. 1997).

Obviously, better understanding and elucidation of metal functioning in processes related to flavor stability, changes during maturation, taste and odor of beer,

Fractionation analysis and metal partitioning pattern

So far, experimental evidence of metal partitioning in beer has been occasionally

reported in the literature. The charge of Cu, Fe and Mn species in bottled beer (Ringnes Pilsner, Norway) was determined using cation (CE) and anion exchange (AE) cartridges combined with off-line ET-AAS measurements [START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF]. When analyzing effluents obtained after passing untreated beer aliquots through separate cartridges, it was found that over 94% of the total Mn is present in the form of positively charged species, likely simple Mn(II) cations. Fe was determined to be completely present as negatively charged species, e.g. Fe bound to different classes of organic compounds. Positively (37%) and negatively (72%) charged species were found for Cu also supposing the existence of different metal complexes. The analysis of beer aliquots spiked with free Cu(II), Fe(III) and Mn(II) indicated that beer contains endogenous ligands that readily bind Cu(II) and Fe(III) ions, forming negatively charged metal complexes. Only added Mn(II) was totally recovered as positive ions.

In the cited work by [START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF], untreated beer was also treated with a size exclusion column so as to assess possible associations of Cu, Fe and Mn with ligands of different molecular mass ranges. The size exclusion chromatograms, constructed on the basis of metal quantities found in column effluents, proved that Mn in beer is present in non-complexed forms, presumably as Mn 2+ . The retention volumes of eluted Cu and Fe species suggested that these metals are likely to be negatively charged species bound to polyphenolic compounds, which molecular mass ranges from 4 to 12 kDa (Cu) and from 4 to 9 kDa (Fe).

A strong CE resin Dowex 50Wx4 was applied by [START_REF] Pohl | Pre-concentration of Cd, Co, Cu, Ni and Zn using different offline ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection[END_REF] to evaluate the cationic fraction for Al, Ba, Cu, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb Sr and Zn in canned beer (Warka Full Light, Poland). It was presumed that the cationic fraction is highly bioavailable to human organisms through gastrointestinal system and comprises free metal cations, stable cationic complexes of metals with small molecular mass, non-volatile acids, e.g. pyruvic acid, malic acid, lactic acid, citric acid, oxalic acid, and labile metal species, dissociating on a resin bed. The content of metals in the cationic fraction was determined by passing an untreated beer sample through the column, followed by resin acid digestion and analysis of a resulting digest. It was found that for Al, Co, Cr and Cu, the contribution of the cationic fraction to the total metal content is relatively low and varies from 6% (Cu) to 11% (Co). For Mn, Ni and Zn, the CF was established to range from 23 to 28%. Much higher donation of the cationic fraction (68-100%) was determined for Ca, Ba and Sr, probably because these metals are present in beer as simple metal cations. Contrary, Fe and Pb were found to be completely present in the form of neutral or negatively charged metal complexes.

Metal fractionation protocols based on solid phase column extraction have been recently proposed for Mn and Zn partitioning in Polish canned and bottled beers [START_REF] Pohl | Fractionation analysis of manganese and zinc in beers by means of two sorbent column system and flame atomic absorption spectrometry[END_REF]Pohl 2007b). Distinct Mn and Zn species, i.e. the polyphenolic metal fraction (PF) and the CF, were retained respectively on a Amberlite XAD7 adsorbing resin (1 st column) and a strong CE resin Dowex 50Wx4 (2 nd column) connected in a series [START_REF] Pohl | Fractionation analysis of manganese and zinc in beers by means of two sorbent column system and flame atomic absorption spectrometry[END_REF]. A comparable tandem column assemblage, in which the 18 adsorbing resin was replaced by a weak AE resin Reillex 402 was also described (Pohl 2007b). Metal concentrations in the distinguished fractions were measured using F-AAS after a subsequent elution of the columns. Only in case of the resin Reillex 402, exposing a very strong adsorptive behaviour toward organic substances through hydrogen bonds with phenolic, hydroxyl and carboxyl groups of the dissolved organic matter, Mn and Zn amounts retained on the column were evaluated by analyzing a portion of the effluent incoming the Dowex 50Wx4 column. Additionally, the residual metal fraction (RF) was assessed by determining metal amounts in effluents obtained from passing beer aliquots through the column systems. The schemes of both tandem column assemblages are given in Figure 1.

<Figure 1 near here> With the proposed procedures Mn and Zn are portioned among hydrophobic complexes of metals with high molecular mass polyphenolics and flavonoids (PF), sum of free metal cations and stable cationic metal complexes with low molecular weight species, including amino acids, organic acids being of primary origin (citric, malic) or secondary products of alcoholic fermentation (pyruvic, lactic, ketoglutaric, succinic, citramalic, fumaric) (cationic fraction), and residual metal species, possibly being anionic and/or neutral metal associations either with low molecular mass ligands (RF).

The distribution of Mn and Zn among PF, cationic fraction and RF in some Polish bottled and canned beers is given in Figure 2.

<Figure 2 near here> In comparison to the results previously reported by [START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF], it seems that possible chemical forms and associations of Mn are more differentiated.

As reported before, it was found that the predominant class of Mn species is the cationic fraction with donation to the total Mn concentration changing from less than 50% (nonpasteurized and award-winning beer brands) to 100% (low-priced beers). There is however a considerable contribution of the RF (14-42%), Mn is also present in the form of complexes with polyphenolic substances, as the PF accounts for up to 37% of the total Mn content. The abundance of the CF for Zn is high (27-85%) and also dependent on beer grade. Zn was found to be bound to polyphenols, but the contribution of the PF is low, i.e. from 2% (low-priced beers) to 21-53% (non-pasteurized beers).

Bearing in mind possible metal associations with natural organic substances present in beer [START_REF] Montanari | Organic and phenolic acids in beer[END_REF][START_REF] Cortacero-Ramirez | Analysis of beer components by capillary electrophoretic methods[END_REF][START_REF] Khatib | Application of two-dimensional J-resolved nuclear magnetic resonance spectroscopy to differentiation of beer[END_REF]), the hydrophobic PF can be regarded as hardly bioavailable to humans while the sum of the cationic fraction and the RF can be assumed as highly absorbable.

The metal groupings distinguished through the fractionation protocols described above are operationally defined and relate to a sorption behavior of metal species toward adsorbents and ion exchangers applied in the fractionation schemes (Pohl 2007a). Certainly, with respect to the total content analysis, the described methods enable retrieving the information on the abundance of individual metal fractions in beer and can yield useful knowledge about metal bioavailability, beer safety, authenticity and nutrition.

Conclusions

In view of excellent beer quality assurance, knowledge concerning metal composition in beer and brewing liquors at various steps of beer production is very important to the brewers. Metal ions affect fermentation, maturation and storage of beer and imply its final flavor and colloidal stability. Considering the nutritious value of metals and the effect they have on beer wholesomeness, information on the total metal content in beer is also advantageous to the consumers. Depending on the concentration, various metals may be toxic to human body or essential for health with a profound effect on wellbeing. When using chemometric data analysis and interpretation methods, the determination of metals in beer is useful in characterization and authentication of beer according to type, origin or packaging.

However, actual bioavailability and absorbability of metals and their effect on beer stability and haze formation depend not only on the total concentrations but also on type of distinct physicochemical forms in which they are present in beer. In commonly applied procedures focused on the total metal content determination after previous beer digestion, any information on the occurrence of possible metal species can not be achieved. Usually, methods linking chromatographic separation techniques with sensitive spectrometric detection can be useful for that purpose and helpful for elucidation of metal functions in beer processing and implications on beer quality, flavor and taste stability. In addition, methods stimulating gut and intestinal conditions can be suitable for estimation of metal bioavailability and toxicity to human organisms. Aluminum [START_REF] Sharpe | Content, chemical speciation, and significance of aluminum in beer[END_REF][START_REF] Vela | Detection of aluminum residue in fresh and stored canned beer[END_REF][START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF][START_REF] Vinas | Determination of cadmium, aluminium, and copper in beer and products used in its manufacture by electrothermal atomic absorption spectrometry[END_REF][START_REF] Briggs | Brewing Science and Practice[END_REF][START_REF] Pohl | Pre-concentration of Cd, Co, Cu, Ni and Zn using different offline ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection[END_REF] 0.08-0.18 0.05-1.24 0.05-2.2 0.005-6.50

Acknowledgment

F

Barium [START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF] 0.020-0.032 0.034-0.049 0.014-0.033 0.016-0.068

Cadmium [START_REF] Briggs | Brewing Science and Practice[END_REF] 0.0002-0.020 0.031-0.40 0.095-0.68

Copper [START_REF] Matusiewicz | Methods for improving the sensitivity in atom trapping flame atomic absorption spectrometry: analytical scheme for the direct determination of trace elements in beer[END_REF][START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF][START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF][START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF][START_REF] Vinas | Determination of cadmium, aluminium, and copper in beer and products used in its manufacture by electrothermal atomic absorption spectrometry[END_REF][START_REF] Bellido-Milla | Beer digestions for metal determination by atomic spectrometry and residual organic matter[END_REF][START_REF] Briggs | Brewing Science and Practice[END_REF][START_REF] Pohl | Pre-concentration of Cd, Co, Cu, Ni and Zn using different offline ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection[END_REF][START_REF] Asfaw | Direct analysis of beer by ICP-AES: a very simple method for the determination of Cu, Mn and Fe[END_REF][START_REF] Nascentes | Direct determination of Cu, Mn, Pb, and Zn in beer by thermospray flame furnace atomic absorption spectrometry[END_REF][START_REF] Llobat-Estelles | Detection of bias errors in ETAAS: Determination of copper in beer and wine samples[END_REF] 0.008-0.80 0.032-0.068 0.019-0.80 0.029-0.050 0.029-0.15 0.024-0.080 0.022-0.16

Iron [START_REF] Matusiewicz | Methods for improving the sensitivity in atom trapping flame atomic absorption spectrometry: analytical scheme for the direct determination of trace elements in beer[END_REF][START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF][START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF][START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF][START_REF] Bellido-Milla | Beer digestions for metal determination by atomic spectrometry and residual organic matter[END_REF][START_REF] Briggs | Brewing Science and Practice[END_REF][START_REF] Pohl | Pre-concentration of Cd, Co, Cu, Ni and Zn using different offline ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection[END_REF][START_REF] Asfaw | Direct analysis of beer by ICP-AES: a very simple method for the determination of Cu, Mn and Fe[END_REF] Page Manganese [START_REF] Matusiewicz | Methods for improving the sensitivity in atom trapping flame atomic absorption spectrometry: analytical scheme for the direct determination of trace elements in beer[END_REF][START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF][START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF][START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF][START_REF] Bellido-Milla | Beer digestions for metal determination by atomic spectrometry and residual organic matter[END_REF][START_REF] Briggs | Brewing Science and Practice[END_REF][START_REF] Pohl | Pre-concentration of Cd, Co, Cu, Ni and Zn using different offline ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection[END_REF][START_REF] Asfaw | Direct analysis of beer by ICP-AES: a very simple method for the determination of Cu, Mn and Fe[END_REF][START_REF] Nascentes | Direct determination of Cu, Mn, Pb, and Zn in beer by thermospray flame furnace atomic absorption spectrometry[END_REF] Lead [START_REF] Briggs | Brewing Science and Practice[END_REF][START_REF] Nascentes | Direct determination of Cu, Mn, Pb, and Zn in beer by thermospray flame furnace atomic absorption spectrometry[END_REF] 0.003-0.024 0.001-0.006 0.0008-0.033

Strontium [START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF] 0.23-0.41 0.14-0.30 0.13-0.74 0.13-0.39

Zinc [START_REF] Wagner | Determination of zinc in wort and beer by graphite furnace atomic absorption spectroscopy[END_REF][START_REF] Matusiewicz | Methods for improving the sensitivity in atom trapping flame atomic absorption spectrometry: analytical scheme for the direct determination of trace elements in beer[END_REF][START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Wyrzykowska | Application of ICP sector field MS and principal component analysis for studying interdependences among 23 trace elements in Polish beers[END_REF][START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF][START_REF] Briggs | Brewing Science and Practice[END_REF][START_REF] Nascentes | Direct determination of Cu, Mn, Pb, and Zn in beer by thermospray flame furnace atomic absorption spectrometry[END_REF][START_REF] Pohl | Fractionation analysis of manganese and zinc in beers by means of two sorbent column system and flame atomic absorption spectrometry[END_REF]Pohl 2007b) 0.013-0.064 0.013-1.48 0.004-0.12 0.001-0.98 0.007-0.28

  regulate metal ions availability by formation of complexes of different stability. Certainly, such a reduction of bioavailability in the case of Ca(II),

  metals in brewing liquors and beer is regulated(Baxter and Hughes

  By sample dilution, the formation of a solid deposit on a burner head, or the coverage of a graphite tube by a remaining ash layer is avoided[START_REF] Sharpe | Content, chemical speciation, and significance of aluminum in beer[END_REF][START_REF] Svendsen | Speciation of Cu, Fe and Mn in beer using ion exchange separation and size-exclusion chromatography in combination with electrothermal atomic absorption spectrometry[END_REF][START_REF] Nascentes | Direct determination of Cu, Mn, Pb, and Zn in beer by thermospray flame furnace atomic absorption spectrometry[END_REF].Instead of sample dilution necessary for measurements performed using ET-AAS, Llobat-Estelles et al. (2006) have proposed a mathematical method of beer analysis based on an apparent content curves model enabling the evaluation of bias errors corresponded to any modification of a shape and a position of analyte absorbance profiles. Accordingly, the described method facilitates accurate and reliable measurements of metals in beer notwithstanding the interferences stemming from different organic constituents present in undiluted beer samples.

  of pattern recognition of beer, different chemometric techniques are usually applied to a data matrix referred to mineral content assessed through the analysis of a high number of different beer brands. Typically, principal component analysis (PCA) is used for visualization of the results, while linear discriminant analysis (LDA) and artificial neural networks (ANN) are supervised learning methods, helpful in finding adequate classification rules. Indeed, information on the mineral beer composition including total concentrations of a number of metals, including Ag, Al, Ba, Ca, Cd, Co, Cr, Cu, Fe, Ga, Hg, K, Mg, Mn, Na, Ni, Pb, Rb, Sr, V, Zn, along with respective pattern recognition analysis is suitable for differentiation and classification of beer according to type, origin and container, or kind of raw materials used for brewing.

  and Fe in canned beers are commonly higher than those in bottled beers (Bellido

  the elucidation of possible associations of these metals with different endogenous beer ligands is of special interest and significance in view of brewing

  and nutritional value referred to beer consumption require the knowledge about speciation and/or fractionation forms of metals in beer. In this view, methods enabling classification of the existing metal groupings in beer and assessment of metal fractionation pattern are very essential.

Figure 1 .

 1 Figure 1. Fractionation schemes for classification of Mn and Zn in beer. (a) Amberlite

Figure 2 .-

 2 Figure 2. Fractionation patterns of Mn and Zn in beer (adapted from Pohl and Prusisz

Figure 1 .Figure 2 .

 12 Figure 1. Fractionation schemes for classification of Mn and Zn in beer. (a) Amberlite XAD7-Dowex 50Wx4 and (b) Reillex 402 -Dowex 50Wx4 tandem column systems. 40x28mm (600 x 600 DPI)

  • , H 2 O 2 , OH • ) are crucial to beer staling process because they willingly react with different classes of organic compounds present in beer, including polyphenols, isohumulones, alcohols, aminoacids, fatty acids, iso-α-acids, α-acids and β-acids, permanently changing a sensory beer profile and determining its flavor stability. Apparently, the rate and level of the reactive oxygen species formation is related to Cu and Fe amounts (Cu + /Cu 2+ , Fe 2+ /Fe

3+

), and thus, concentrations of Cu and Fe in beer are of special significance and should be minimized and/or controlled during the brewing process.

Table II .

 II Concentration of major metals in beer of different country of origin.Magnesium[START_REF] Bellido-Milla | Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves[END_REF][START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF][START_REF] Briggs | Brewing Science and Practice[END_REF] Pohl and Sodium (Bellido-Milla et al. 2000;[START_REF] Alcazar | Multivariate characterisation of beers according to their mineral content[END_REF][START_REF] Briggs | Brewing Science and Practice[END_REF] TableIII. Concentration of minor and trace metals in beer of different country of origin.

	Page 31 of 35	Food Additives and Contaminants
		Concentration, mg l -1 British Dutch Calcium (Bellido-Milla et al. 2000; Alcazar et al. 2002; Briggs et al. 2004) German Spanish 40.0-140 42.2-69.8 3.80-108 29.0-86.2 Potassium (Bellido-Milla et al. 2000; Alcazar et al. 2002; Briggs et al. 2004) Others 16.5-111 135-1100 124-648 46.7-833 22.9-496 17.5-442 Prusisz 2004) 60.0-200 55.5-265 23.7-266 42.0-110 50.0-112 21.9-230 10.4-47.6 1.19-120 3.95-103 4.13-66.4 Concentration, mg l -1 F o r P e e r R e v i e w British Dutch German Norwegian Polish O n l y Spanish	Others

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Katerina Lancova

Jana Hajslova

Jan Poustka

Alexandra Krplova

Milena Zachariasova

Pavel Dostálek

Lenka Sachambula

P Dostalek

Sachambula

TRANFSER OF FUSARIUM MYCOTOXINS AND "MASKED" DEOXYNIVALENOL (DEOXYNIVALENOL-3-GLUCOSIDE) FROM FIELD BARLEY THROUGH MALT TO BEER

Keywords: Deoxynivalenol, deoxynivalenol-3-glucoside, conjugated mycotoxins, Fusarium mycotoxins, 16 barley, malt, beer, processing. 17 18

published or not. The documents may come

INTRODUCTION 19

Beer contributes significantly to the diet of the population world-wide. The British 20

Beer and Pub Association has reported recently (2006) that the annual per-capita consumption 21 of this fermented drink in 2004 exceeded 100 litres in many countries, with a maximum of 22 157 litres in the Czech Republic. Unfortunately, barley, the major raw material used in beer 23 production, like other cereals, can often be infected under field conditions by toxinogenic 24 Fusarium species, causing the disease called Fusarium head blight and, consequently, 25 and aroma components to beer, can be also contaminated with Fusarium Spp. (Solarska 2 2007), the disease is called in this case Fusarium cone tip blight [START_REF] Bienapfl | Fusarium cone tip blight: A new disease 5 of Humulus Lupulus[END_REF]. [START_REF] Payen | Sur la presence de mycotoxines dans des 4 bieres[END_REF] However, until now hops have seldom been investigated as a possible source of Fusarium 4 mycotoxins [START_REF] Hazel | Influence of processing on trichothecene levels[END_REF]. [START_REF] Ruprich | Determination of the mycotoxin deoxynivalenol in beer by 6 commercial ELISA tests and estimation of the exposure dose from beer for the population in 7 the Czech Republic[END_REF] In 2003, a report on task for Scientific Cooperation (SCOOP) concerned with 6 occurrence of Fusarium toxins in a wide range of food commodities, the data needed for 7 assessment of dietary intake by the population of EU Member States become available 8 (http://ec.europa.eu/food/fs/scoop/index_en.html). The selected information on the occurrence 9 of these mycotoxins in barley and malt reported by participating countries (Finland, France, 10 Italy, The Netherlands, Norway, UK) is summarised in Table I. As documented here, DON,[START_REF] Scott | Analysis of Canadian and imported beers for 12 Fusarium mycotoxins by gas chromatography[END_REF] the representative of trichothecenes B group, is the most abundant Fusarium toxin in these 12 key raw materials used for beer production. The extent of its transfer into beer analysed in 13 various countries worldwide is shown in Table II [START_REF] Omurtag | Occurrence of deoxynivalenol (vomitoxin) in beer in 22 Turkey detected by HPLC[END_REF], Papadopoulou-14 [START_REF] Bouraoui | Screening 1 survey of deoxynivalenol in beer from the European market by an enzyme-linked 2 immunosorbent assay[END_REF][START_REF] Odhav | Mycotoxins in South African traditionally brewed beer[END_REF][START_REF] Molto | Occurrence of trichothecenes in 14 Argentinean beer: a preliminary exposure assessment[END_REF], Shim et al. 1997, Ruprich 15 and Ostry 1995, Weddeling et al. 1994, Niessen and Donhauser 1993, [START_REF] Scott | Analysis of Canadian and imported beers for 12 Fusarium mycotoxins by gas chromatography[END_REF], 16 Trucksess et al. 1986[START_REF] Payen | Sur la presence de mycotoxines dans des 4 bieres[END_REF]). In the two most extensive available studies focused 17 on DON behaviour during malting and/or brewing processes [START_REF] Schwarz | Fate and Development of Naturally Occurring 9 Fusarium Mycotoxins During Malting and Brewing[END_REF], Niessen et 18 al. 1993), fairly contradictory results, both DON increase and its decrease as compared to [START_REF] Odhav | Mycotoxins in South African traditionally brewed beer[END_REF] barley used for processing, were found, see Table III. Besides DON, the presence of ZON and 20 15-ADON was found in malts employed for brewing experiments, however, in the later 21 stages, their occurrence above the method LOD, was documented only in spent grains, not in 22 beers [START_REF] Schwarz | Fate and Development of Naturally Occurring 9 Fusarium Mycotoxins During Malting and Brewing[END_REF]. It should be noted, that ZON is metabolised to αand β-23 zearalenol by brewing strains of Saccharomyces cerevisiae. However, the occurrence of ZON 24 and its metabolites in beer was reported only rarely, Zöllner at el. (2000) detected these 25 different grains and under different brewing conditions. Overall, no more information on the 2 fate of Fusarium toxins, others than DON, 15-ADON and ZON, during the malting and 3 brewing processes has been available until now. Also the occurrence of conjugated forms of 4 mycotoxins in beer has previously not been investigated, although their presence in cereals is 5 known. The first report on "masked" mycotoxins represented typically by toxin bound to a 6 more polar molecule like glucose, amino acids, or sulphate, appeared in the mid of the 80s 7 when the severity of mycotoxicoses in animals was found to be greater than their determined 8 levels in feedstuffs. It was suggested, that hydrolysis of toxin conjugates occurs in the 9 digestive tracts releasing the levels of precursor toxins, thus increasing animals' exposure 10 (Gareis 1994). Later on [START_REF] Berthiller | Masked 1 Mycotoxins: Determination of a Deoxynivalenol Glucoside in Artificially and Naturally 2 Contaminated Wheat by Liquid Chromatography-Tandem Mass Spectrometry[END_REF], deoxynivalenol-3-glucoside (DON-3-Glc) was 11 reported as the major form of "masked" DON constituting up to 12% of total content of this 12 mycotoxin in examined cereals, wheat and maize. 13

To avoid consumers' health risk due to unacceptably high dietary intake of Fusarium standard on to the market allowed the LC-MS/MS method to be established for this analyte in 1 the matrices collected within the current malting and brewing experiments. In this way, it was 2 possible for the first time to obtain information on the possible transfer of "masked" 3 mycotoxins from barley infected by fusaria into beer. 4

5

MATERIALS AND METHODS 6

Chemicals 7

Standards of analysed mycotoxins (deoxynivalenol, DON; deoxynivalenol-3-8 glucoside, DON-3Glc; nivalenol, NIV; fusarenon-X, FUS-X; sum of 15-and 3-acetyl-9 deoxynivalenol, ADONs; T-2 toxin, T-2; HT-2 toxin, HT-2 and zearalenone, ZON) were 10 purchased from Biopure (Austria). To ensure the accuracy of measurements certified 11 reference material, DON naturally contaminated wheat (R-Biopharm, UK), ZON naturally 12 contaminated milo (R-Biopharm, UK) and DON in wheat flour (BCR, Belgium), were used. [START_REF] Molto | Occurrence of trichothecenes in 14 Argentinean beer: a preliminary exposure assessment[END_REF] The BCR reference materials were produced and certified in accordance with internationally 14 accepted rules laid down in the various guides by BCR (Bureau Communautaire de 15 Référence), the ISO (International Organisation for Standardisation) and the WHO (World 16 Health Organisation). 17

Samples 18

The levels of Fusarium toxins in barley and malt grists that were used for processing 19 experiments are summarized in Table IV of processing experiments, mycotoxin levels in malt grists taken for brewing were not 9 identical with those determined in products obtained in malting. 10

Processed products 11

Malted barley 12

Micro-malting experiments that were carried out by the Malting Institute of the 13 Research Institute of Brewing and Malting (Brno, Czech Rep.) in line with traditional 14 processes. Barley grains (3.5 kg) were micromalted in s small-scale malting equipment of the 15 company KVM (CR). Malt was produced from barley within 3 key steps: i) steeping, ii) 16 germination and iii) kilning. To initiate germination, the barley must be adequately supplied 17 with both water and oxygen during a steeping stage and this was achieved in this study. The 18 production of enzymes is the main purpose of the germination step. During the kilning stage, [START_REF] Odhav | Mycotoxins in South African traditionally brewed beer[END_REF] water is removed from the green malt and the malt then becomes stable and storable. Detailed 20 descriptions of the experimental malting conditions and a list of samples taken during this 21 process are summarised in Table V. [START_REF] Solarska | Study on cause of Fusarium cone tip blight[END_REF] Beer 23 Beer was produced from malt grist according to a standard procedure in the brewery 24 pilot plant at the Department of Fermentation Chemistry and Bioengineering (Institute of 25 Chemical Technology -ICT, Prague). In brief, the malt grist was mashed in a double mash 1 process. After mashing, the liquid portion was separated from the spent grains by the classical 2 lautering process. Then the wort was boiled together with added hop. After hot trub 3 separation, fermentation was carried out with bottom-fermenting yeast. Detailed experimental 4 brewing conditions together with a description of the samples taken throughout the beer 5 production is summarised in Table VI. 6 7

Mycotoxin analysis 8

Sampling 9 500 g of grains were taken from thoroughly mixed barley batches delivered to the 10 laboratory. These representative samples were ground in the laboratory mill (LM 120; Perten 11 Instruments). For mycotoxin analysis, two sub-samples (each 12.5 g) were taken. Similar 12 approach was employed for sampling of representative malt grists and other processing 13 intermediates. Regarding liquid brewing intermediates and beers, 16 ml aliquots used for 14 mycotoxin analysis were taken from100 ml samples collected within process. 15 16 Extraction and clean-up procedure for solid samples 17

Representative samples (12.5 g) were extracted with 50 ml acetonitrile:water mixture 18 (84:16,v/v) for one hour using an automatic shaker (IKA Laboratortechnik, Germany). The 19 crude extracts were then filtered (Filtrak No. 390, VEB Freiberger, Germany) and 8 ml 20 aliquots transferred into sample tubes to which 80 µl of acetic acid (99%, Sigma-Aldrich) 21 were added. Purification was achieved by solid phase extraction (SPE) employing 22

MycoSep TM 226 cartridges (Romer, Austria). 4 ml of purified extract were evaporated to 23 dryness and the solid residues transferred into 1 ml of a water: methanol mixture (50:50, v/v), 24 and passed through a 0.2 µm microfilter (Alltech, USA) prior to further analysis. [START_REF] Berthiller | Masked 1 Mycotoxins: Determination of a Deoxynivalenol Glucoside in Artificially and Naturally 2 Contaminated Wheat by Liquid Chromatography-Tandem Mass Spectrometry[END_REF] Extraction and clean-up procedure for liquid samples 26 The aliquots (16 ml) of processing intermediates (sweet wort, warm wort, wort after 1 trub removal, green beer) as well as the final product (beer), were shaken after the addition of 2 84 ml acetonitrile and 3.2 g Celite for one hour using an automatic shaker. The extract 3 obtained was then filtered and 5 ml aliquots were evaporated to dryness. The residues were 4 transferred into 1 ml of a water: methanol mixture (50:50, v/v) prior to the analysis. 5

High performance liquid chromatography coupled with tandem mass spectrometry 6 (LC-MS/MS) 7

High performance liquid chromatography (HP1100 Binary Series LC system, Agilent 8

Technologies, USA) coupled with mass spectrometer (Finnigan LCQ Deca, USA) were used 9 for the analysis. Chromatographic separation of sample components was carried out on a 10 reverse phase column with polar end-capping (Synergi Hydro RP, 150mm x 3mm x 4µm, 11 Phenomenex, USA) held at 40 ºC and operated under gradient conditions. The mobile phase 12 was composed of 10 mM amonium acetate in purified water (A) and methanol (B). The flow 13 rate of the mobile phase was set to 0.5 ml/min and the injection volume was 20 µl. Gradient 14 elution was performed starting from A:B (80:20, v/v) and reaching A:B (30:70, v/v) in 8 min. 15

From 8 to 15 min the ratio A:B (30:70, v/v) was stable and then jumped to A:B (80:20, v:v). 16 The time of post run lasted 7min. 17 Performance characteristics of analytical method / Quality assurance 5 Limits of detection (LODs) and quantification (LOQs), recoveries and repeatabilities 6 expressed as RSDs that were obtained within the validation process are reported in Table VII. [START_REF] Hazel | Influence of processing on trichothecene levels[END_REF] Calibration curves for all analytes were linear within the working range from 5 to 5000 µg/kg. 8

Squared correlation coefficients (R 2 ) were under 0.999 for 10 point calibration curves. The 9 analyses of all examined samples were performed in duplicate. The data shown in Tables VIII 10 and IX are mean values corrected for analytes recoveries. 11

The method used for samples analysis of was accredited (ISO 17025) for cereals and, 12 as a part of external quality control, the trueness of generated data was successfully 13 demonstrated through participation in proficiency testing Food Analysis Performance 14 Assessment Scheme (FAPAS) organized by Central Science Laboratory (CSL, York UK). 15 the key raw material for beer production. To estimate consumers' exposure to Fusarium 2 toxins from this commodity and to support identification of measures aimed at its reduction 3 (as in the case of other contaminants), the knowledge on the fate of these hazardous fungal 4 secondary metabolites through the production of beer is essential. The currently available 5 information on the changes of DON and other Fusarium toxins during barley processing is 6 rather contradictory and of new interest is none of the available studies was concerned with 7 "masked" mycotoxins issue. The results obtained within this research on the behaviour of 8

Fusarium toxins both during malting and brewing addresses some of these issues and should 9 contribute to a better understanding of their fate during the beer making process. 10 11

Malting process 12

It is well established that, barley, depending on a crop year and agricultural practices 13 employed, is often to some extent infected by Fusarium fungi, and consequently by their toxic 14 VIII. Although DON-3-Glc, ADONs, HT-2 were below the detection limits in the NAT 5 barley sample, these mycotoxins were detected in this germination stage. The experimental 6 growth of Fusarium toxins shown in Figure 1 and Table VIII might be associated with a rapid 7 onset of enzymatic activities in the germination of barley kernels, alternatively, de novo 8 formation of fungal secondary metabolites is another conceivable cause. Worth to notice, that 9 not only increase, but also reduction of DON in malt as compared to barley used for its 10 production was reported in one of few older studies [START_REF] Schwarz | Fate and Development of Naturally Occurring 9 Fusarium Mycotoxins During Malting and Brewing[END_REF]. 11

The final malting stage, kilning, did not change "free" trichothecenes levels 12 significantly, neither thermodegradation (Fusarium mycotoxins are stable up to 120°C), or 13 their additional formation as fungal growth was halted by the high temperature. The trend for 14 DON-3-Glc was slightly different in the samples series NAT compared with ART, see Table 15 VIII. While its levels dropped in the samples NAT, no distinct changes were observed in 16 series ART and information on DON-3-Glc stability at higher temperatures is not clear from 17 these data. [START_REF]Collection of occurrence data of 19 Fusarium toxins in food and assessment of dietary intake by the population of EU Member 20 States[END_REF] The final barley malt was obtained by removing the rootlets. It should be noted, that 19 this waste product contained the highest mycotoxin levels of all intermediates taken during 20 malting process (see Table VIII). Considering its use as a nutritive component of feeding 21 stuffs and/or "healthy" food supplements exposure resulting from rootlets by both humans 22 and farm animals should be taken into consideration. 23

Regarding the comparison of Fusarium toxins in the raw material (barley, series ART) 24 and the final product (malt), the levels of DON, DON-3-Glc, ADONs and HT-2 were 2.1, 8.6, 25 1.1 and 2.1 times higher in malt than in barley, respectively, see Figure 1. In series NAT, 1 DON was the only mycotoxin detected in barley, nevertheless, the malt prepared from this 2 sample contained all the spectrum of mycotoxins (although at lower levels) as that found in 3 ART. DON increased 3.8 times. The release from conjugated forms and/or their de novo 4 production by growing fungi is likely to be the cause of trichothecenes levels being elevated 5 during the malting process. 6 7

Brewing process 8

The ingredients needed for beer production are malt grist, hops, yeast and water. 9

Whereas the last two items are hardly the source of mycotoxins, malt grist, as shown above, 10 may supply significant amounts of these toxins to beer. Theoretically, hops may also contain 11 toxic secondary metabolites produced by fungi, but no information suggesting this commodity 12 as a source of contamination is available. In any case, hop extract is largely diluted to beer 13 (3.4 g of hops per 1 l of beer) so their likely contribution to the overall load of Fusarium 14 toxins is likely to be low. Within this part of the study, two series of malt grists prepared from 15 NAT and ART together with respective final beers, were analysed for 7 trichothecenes and 16 ZON; and the levels of "masked" DON, DON-3-Glc, were also determined. The results 17 obtained are summarised in Table IX, and illustrated in Figures 2 and3, in which the transfer 18 of DON, DON-3-Glc, ADONs and HT-2 was calculated as a percentage according to the 19 mass balance from the starting raw material (malt grist; 6 kg) into final beer (50 l). For better 20 understanding of mass balance calculation based on data shown in Table VI, an example of 21 DON-3-Glc transfer from malt grist to sweet wort follows: conjugate concentration in the 22 NAT malt grist was 349 µg/kg giving a total amount of 2,094 µg in the 6 kg needed for 23 preparation of 59 l sweet wort, in that DON-3-Glc level was 561 µg/kg giving a total amount 24 As expected, most of DON was removed from malt grist to beer, this toxin was not 3 detected (LOQ = 5 µg/kg) in NAT spent grains (the waste from mashing step) and only 1% of 4 the total DON was found in more contaminated ART sample. DON transfers to sweet wort 5 were 70 and 67% in NAT and ART series, respectively. In hot wort and wort after trub 6 removal corresponding to the ART series, DON transfer exceeded even 100%. In green beer 7 as well as in the final beer, the total content of DON in both sample series, NAT and ART, 8 was lower as compared to its total content in the malt grist batch taken for brewing 9 experiments. These results are in a good agreement with an earlier study by Schwarz et al. 10 (1995), who reported similar DON transfer rates where the values were in the range of 80-11 93% in four parallel brewing experiments resulting from highly contaminated malt grists (see 12

Table III in Introduction). A distinctly opposite trend, huge increases of DON in beers, as 13

high as 300 and 600%, were reported by [START_REF] Niessen | Fate of deoxynivalenol in the process of brewing and its 17 prevalence in commercial beer[END_REF], who used two malt 14 grists with similar DON levels (see Table III) as those in the current study. It should be noted; 15 that a relatively accurate method (although not enabling low detection limits), gas 16 chromatography coupled with mass spectrometry (GC/MS), was used by Schwarz et al. 17 (1995) for target analytes quantification whereas a screening enzyme-linked 18 immunoabsorbent assay (ELISA) based on a polyclonal antibody was employed in the [START_REF] Odhav | Mycotoxins in South African traditionally brewed beer[END_REF] Niessen's and Donhauser's study. Large discrepancies between the results of these studies can 20 be due to possible cross-reactions associated with the ELISA method. Fairly beyond 21 expectation, the content of this "masked" DON increased dramatically in sweet wort, hot wort 22 and wort after trub removal prepared from both sample series, NAT and ART, as compared to 23 its level in malt grist taken for processing. In the later case, DON transfer in these 24 intermediates exceeded even 10 3 %. The dynamics of DON-3-Glc was rather different 25 between the two experimental series; nevertheless, in terms of transfer to the final product, it 1 was equal at 600% in both beers derived from NAT and ART malt grists. Interestingly, this 2 value closely corresponded to "DON increase" reported by [START_REF] Niessen | Fate of deoxynivalenol in the process of brewing and its 17 prevalence in commercial beer[END_REF], 3 see Table III. Based on preliminary tests, for which several types of commercial ELISA kits 4 were employed, we showed the cross-reactivity towards DON-3-Glc to be comparable to that 5 of DON. With regard to this, until now not reported observation, it can be assumed that the 6 data reported in Niessen's study were significantly overestimated due to the presence of DON 7 conjugate. Its hydrolysis from bounds to higher molecular constituent is probably supported 8 by high temperatures, 70 and 100°C, occurring in sweet wort and hot wort technological 9 stages. To get more knowledge on "masked" mycotoxin transformation, we have initiated 10 intensive research in this area . 11 In effect, ADONs, represent, another form of masked DON. As shown in Figure 2 and 12 3, their levels in experimental samples were almost equal or slightly higher as compared to 13 DON. Transfer into final beers were slightly above 100%. Unfortunately, under the 14 chromatographic conditions employed in our method (several reversed phase columns were 15 tested), we were not able to separate 3-and 15-acetyldeoxynivalenol. It should be noted that 16 in most of commercial DON ELISA kits, high cross-reactivity (sometimes even higher than 17 100%) towards 3-acetyl DON is occur is declared by producers (no declaration for DON-3-18 Glc). Theoretically, this trichothecene could also contribute to overestimated data shown in 19 the study by [START_REF] Niessen | Fate of deoxynivalenol in the process of brewing and its 17 prevalence in commercial beer[END_REF]. 20

As shown in Table VI, series ART, ZON was also transferred into beer and this 21 estrogenic mycotoxin can be largely metabolised by Saccharomyces cerevisiae to β-22 zearalenol. However, this component was not monitored in our experiments. Also considering 23 the possible occurrence of zearalenon-4-glucoside (ZON-4-Glc) in cereals (Engelhardt et al., 24 1999), it is difficult to assess the apparent ZON transfer at 80%, since both its transformation 25 This study concerned with the fate of Fusarium toxins in the beer production chain 5 starting from barley has shown many interesting results of which some have never been 6 reported before. 7

Significant increases of Fusarium toxins (DON, ADONs and HT-2) occurred during 8 malting process, mainly in the germination step. In addition to these trichothecenes, the 9 presence and formation of high levels of DON-3-Glc was documented. Further significant 10 increases in levels of this DON conjugate occurred during the brewing process. In sweet wort 11 its relative content was ten times higher as compared to that in malt grist taken for the 12 processing experiment. Generally, two different phenomena should be considered when 13 explaining high levels of DON-3-Glc that can be found in beer: 14

(i)
De novo growth of Fusarium micromycetes (either coming from spores or 15 hyphae invading the kernels, or due to cross-contamination during this process) 16 favoured under certain malting conditions (3 days, 14.5°C, moisture 45%) is 17 accompanied by production of additional mycotoxins and their transformation 18

products. 19 (ii)

Enzymes produced during the mashing of malt grist degrade cell walls, 20 membrane bound proteins, and starch depots in kernels, thus releasing DON-3-Glc 21 from insoluble forms. [START_REF] Solarska | Study on cause of Fusarium cone tip blight[END_REF] To get a deeper knowledge on the effect of processing on Fusarium toxins and their 23 "masked" forms, more research is needed. Besides beer making other processing practices, 24 namely those employing fermentation such as bread making, should also be investigated to 25 identify both additional risk sources as well as possibilities to reduce the total mycotoxin load 1 in foods. Although the bio-availability of mycotoxin conjugates has not been fully 2 documented yet, the possibility of approaching, or even exceeding TDI of 1 µg/kg body 3 weight of DON established by Scientific Committee on Food (SCF) (European Commission 4 2006), could occur. By supposing similar toxicological profile for DON and DON-3-Glc, 5 these conditions could occur for a 60 kg person when drinking approx. 2 glasses of 6 "experimental" beer prepared in our study from naturally infected barley in which the sum of 7 both "free" and conjugated DON was 77 µg/l. 8

There are discrepancies between the data in the literature on DON content in the beer 9 production chain obtained by ELISA, and our results which were established by LC-MS/MS. 10 This and other preliminary work at our laboratory lead us to assume that in part may be due to 11 cross-reactivity of commercial test kits towards DON-3-Glc and other forms of DON. In any 12 case, challenges in mycotoxins analysis, including the possibility to employ immunoafinity 13 columns for simultaneous pre-concentration of both "free" and "masked" mycotoxins, have 14

  by mycotoxins. It should be noted, that hops, an ingredient supplying bittering 1

  one of 23 beer samples collected in different countries, produced from 1

  14 toxins, the European Commission has established the maximum levels for DON (1250 µg/kg 15 in cereals; 200 µg/kg in processed cereal-based foods and baby foods) and ZON (100 µg/kg in 16 cereals; 20 µg/kg in processed cereal-based foods and baby foods). The regulation for other 17 fusariotoxins, the sum of HT-2 and T-2 toxin, is currently under preparation and is expected 18 to come into the force in July 2008 (European Commission 2006). The temporary tolerable 19 daily intakes (TDI) 1, 0.7, 0.2, and 0.06 µg/kg b.w. have been proposed for DON, NIV, ZON 20 and the sum of T-2 and HT-2, respectively (European Commission 2006). However, it does 21not appear that the occurrence and significance of "masked" mycotoxins has been considered 22 in the formulation of these limits. 23The study reported here has investigated the fate of Fusarium mycotoxins within all 24 steps involved in the beer production chain. The recent introduction of a pure DON-3-Glc 25

  Identification and quantification of analytes was performed using a tandem in time mass 18 spectrometry (MS/MS) realised by ion trap with the following parameters (ion source type -19 APCI operated both in negative and positive ion modes under following conditions: capillary 20 temperature -150 °C, vaporizer temperature -450 °C, nitrogen sheath gas flow -1.2 l/min, 21 nitrogen auxiliary gas flow -3 l/min, source voltage -6 kV, collision gas -helium, scan type -22 selected reaction monitoring). APCI positive ions in form [M+NH 4 ] + were used for type A 23 trichothecenes. The ion transitions: parent > daughters (quantifier-Q, identifier-I) were as 24 follows: HT-2: 442>273(Q),299(I) and T-2: 488>305(Q),245(I). For the type B 25 fragmentation of [M+CH 3 COO] -in case of NIV: 371>311(Q),281(I); 1 DON: 355>295(Q),265(I); DON-3-Glc: 517>457(Q),427(I); ADON: 397>337(Q),307(I); 2 FUS-X: 413>353(Q),263 (I) and [M-H] -in the case of ZON: 317>273(Q),299(I) were 3 performed. 4

  The z-scores for analyses of HT-2 and T-2 in oat (Proficiency Test (PT) 2234), ZON in baby 16 food (PT 2236) and maize (PT 2219), DON in wheat flour (PT 2229) and maize (PT 2224) 17 were in the range ±2. 18 19 Moisture determination 20 Moisture content was determined gravimetrically by drying samples in an oven for 2 21 hours at 131 ± 2 ºC according to ISO Standard No. 712 "Moisture determination in cereals 22 and cereal products"Fusarium mycotoxins are almost unavoidable natural contaminants of barley which is 1

  secondary metabolites. The dynamics of Fusarium toxins documented by the analysis of 10 15 samples collected during the malting of both naturally (NAT) and artificially (ART) 16 contaminated barleys shown in TableVIII. The relative transfer of DON, DON-3-Glc, 17ADONs and HT-2 (when taking their content in processed barley grains equal to 100%) are 18 illustrated for sample series ART in Figure1.[START_REF] Odhav | Mycotoxins in South African traditionally brewed beer[END_REF] During the steeping stage, DON as well as ADONs levels were either eliminated 20 (series NAT) or largely reduced to below 10% (series ART) of their original amount in the 21 sample of barley. Similarly, a decline of DON in samples examined during this malting step, 22 was earlier observed by[START_REF] Schwarz | Fate and Development of Naturally Occurring 9 Fusarium Mycotoxins During Malting and Brewing[END_REF]. Interestingly, there was a slight increase of 23 DON-3-Glc in sample series ART occurred within steeping. While "free" mycotoxins were 24 obviously extracted by steep water, the removal of DON conjugate, probably due to its release 25 polysaccharides, was not complete. Further work is required to examine the processes 1 involved with the DON conjugate at this and other processing stages. 2During the germination stage, significant increases of mycotoxin concentrations in 3 processing intermediates occurred in both series of experiments (NAT and ART), see Table4

  441 µg. Employing these figures for "transfer" of DON-3-Glc from the raw material to 1 the first brewing intermediate was 29,441 / 2,094 = 14.05. 2

  elimination may be due to it being bound and released from conjugates during brewing

  research was supported by the international project Truefood (FOOD-CZ-2006-18 016264). Part of funding was obtained from project MSM 6046137305 granted by the 19 Ministry of Education, Youth and Sports of the Czech Republic. Thanks for provision of 20 barley samples belong to Marie Vanova MSc. from the Agrotest Fyto Ltd. (Kromeriz, Czech 21 Rep.). The ideas obtained within intensive discussion with Dr. Berthiller (IFA-Tulln, Austria) 22 are incorporated in conclusions. Authors wish to express appreciation of his scientific input. 23 Trucksess MW, Flood MT, Page SW. 1986. Thin layer chromatographic determination of 1 deoxynivalenol in processed grain products. Journal of Agricultural and Food Chemistry 69: 2 35-36. 3 Weddeling HMS, Babler H, Doerk H, Baron G. 1994. Orientierenden versuche zur 4 anwendbarkeit enzymimmunologischer verfahren zum nachweis von deoxynivalenol, 5 ochratoxin A und zearalenon in braugerste, malz une bier. Monatsschrift fur Brauwissenschaft 6 3: 94-98. 7 Zöllner P., Berner D., Jodlbauer J., Lindner W. 2000. Determination of zearalenone and its 8 metabolites αand β-zearalenol in beer samples by high-performance liquid chromatography-9 tandem mass spectrometry. Journal of Chromatography B 738, 233-241

Figure 1

 1 Figure1Transfer of DON, DON-3-Glc, ADONs and HT-2 from barley to malt, sample series ART (total amount of mycotoxins in process batch of barley grains = 100%; contents of these mycotoxins in analysed samples are shown in TableVIII; sample codes are specified in TableV)

Table I

 I Survey of Fusarium toxins in barley and malt in some EU countries; P/T = number of positive samples/total number of examined samples; mean = mean concentration (µg/kg) calculated from positive samples; max = maximum concentration (µg/kg) (Scientific CooperationReport, 2003) Table II Survey of DON levels (µg/l) in beers as reported by countries worldwide; mean was calculated from positive samples TableIIIOverview of literature data on changes of DON during barley processing, malting and brewing; in parenthesis DON (mg/kg) in raw material -barley)

	Page 21 of 27	Food Additives and Contaminants Food Additives and Contaminants	Page 22 of 27
	*this information was not available Country Food Commodity P/T Country Incidence Process Sample barley 46/77 Finland malt 28/68 barley 9/9 France malt 185/350 Italy barley * Netherlands barley 5/40 Norway barley 6/20 United Kingdom barley 26/153 F o r P e e r R e v i e w DON NIV 3-ADON T-2 Mean Max P/T Mean Range Reference % Remaining DON in processing intermediates and products Mean Max P/T Mean Max P/T Mean Max P/T Mean Max P/T Mean Max HT-2 ZON 111 619 4/77 * 351 6/77 * 101 0/77 * * 3/77 23 41 6/77 18 53 106 394 21/68 135 293 0/68 * * 5/68 58 153 13/68 45 47 0/50 * * 15 35 9/9 * 40 0/9 * * 0/9 * * 0/9 * * * * * * 550 0/194 * * 0/194 * * 0/194 * * 0/194 * * * * * * * * * * * * * 1/1 280 280 * * * 1/1 5 5 299 510 * * * * * * * * * * * * * * * 47 60 0/20 * * 0/20 * * * * * * * * 1/20 11 11 12 53 10/153 25 109 9/153 7 37 0/153 * * 0/153 * * * * * O n l y Afrika 0/35 * * Odhav and Naicker 2002 Argentina 22/50 14.3 4 -221 100 (10.6) 100 (22.5) 100 (8.5) 100 (4.8) 100 (<0.2) * * Barley Molto et al. 2000 Canada ** 29/50 5.4 0.3-50 Steep-out 10 11 0 0 0 * * Scott et al. 1993 Czech Republic 59/77 13 7-70 Green Malt 36 55 18 115 0 * * Malting Ruprich and Ostry 1995 France 1/49 20 * Payen et al. 1983 Germany 85/196 205 up to 569 Niessen and Donhauser 1993 Germany 18/18 5 up to 9 Weddeling et al. 1994 Korea 14/54 * 1-23 Shim et al. 1997 USA 0/14 * * Trucksess et al. 1986 Turkey 0/50 * * Omurtag et al. 2007 Austria 25/33 10.2 4.5-29.5 Belgium 47/47 18.1 4.1-56.7 Cyprus 5/6 8 4-12.2 Czech Republic 17/17 21.5 4.6-55.3 Denmark 9/9 19.9 6-47.1 France 24/27 11 4.1-30.2 Finland 3/4 7.4 5.2-10.6 Germany 45/46 4.7 4-40.5 Greece 4/4 17 16.2-16.8 Hungary 2/2 10.8 10.5-11.1 Ireland 2/2 8.7 7.7-9.6 Italy 16/16 10.5 5-29.4 Netherlands 3/4 8 5.9-9.7 Norway 3/4 7.7 6-9.9 Poland 10/10 17.2 5-32.9 Sweeden 4/7 5.1 5.1-14.6 Slovakia 9/12 13.5 5.5-36.9 Spain 6/13 7.3 5.1-12.2 United Kingdom 25/33 10.9 4.1-30.8 Papadopoulou-Bouraoui et al. 2004 * this information was not available ** including 17 imports O n l y Kilned Malt 33 55 16 100 0 * * Malt Rootlets 48 225 46 300 0 * * Malt Grist 100 (3.5) 100 (17.3) 100 (1.8) 100 (6.2) * 100 (0.44) 100 (0.034) Mash, 50°C * * * * * 266 500 Mash, 62°C * * * * * 320 529 Mash, 72°C * * * * * 405 765 First Wort * * * * * 234 588 Sparging * * * * * 157 441 Kettle full Wort * * * * * 384 735 Last Sparging * * * * * 23 0 Spent Grains 3.4 1.8 0.0 0.0 * 0 0 Hot Wort * * * * * 364 794 Fermentation, day 2 * * * * * 355 * Fermentation, day 6 * * * * * 2625 * Fermentation, day 14 * * * * * 309 588 Brewing Beer 93.4 85.0 90.0 80.0 * 323 588 Reference Schwarz et al. 1995 Niessen and Donhauser 1993 F o r P e e r R e v i e w *this information was not available

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Table IV

 IV Fusarium toxins* (µg/kg dry weight) in barley grains and malt grist used for processing experiments

	Commodity	Sample Code	Fusarium Infection	Sample Series Code	DON	DON-3-Glc	Mycotoxin ADONs HT-2 ZON
	Barley Grains	BG	natural artificial	NAT ART	12 238	< 5 14	< 10 140	< 10 11	< 5 < 5
	Malt Grist	MG	natural artificial	ART NAT	316 1712	67 349	133 933	< 10 < 10	< 5 77
	* NIV, FUS-X and T-2 were not detected in any analysed samples

Table VIII

 VIII Fusarium toxins* (µg/kg dry weight) in samples collected during malting process (Sample codes are shown in TableV) FUS-X, T-2 and ZON were not detected in any analysed samples Table IX Fusarium toxins* in samples collected during brewing process (Sample codes are shown in Table VI) FUS-X and T-2 were not detected in any analysed samples

	Page 25 of 27								Food Additives and Contaminants
	KII M R	44 45 553	10 10 460	20 17 571	32 17 1061	540 510 1067	109 125 1911	228 160 641	33 22 493		Formatted: Font: 8 pt Formatted: Font: 8 pt
	* NIV, Sample Series NAT Sample Unit DON-Code DON 3-Glc ADONs HT-2 ZON	DON	Sample Series ART DON-3-Glc ADONs HT-2 ZON		Formatted: Font: 8 pt Formatted: Font: 8 pt Formatted: Font: 8 pt
	Sample Series NAT DON-3-Glc ADONs HT-2 DON <5 <10 <10 238 <5 <10 <10 27 <5 <10 <10 21 <5 <10 <10 24 16 <10 <10 105 52 19 16 204 MG µg/kg 316 Sample Code DON BG 12 SI <5 SII <5 SIII <5 GI 11 GII 21 67 133 < 10 SG µg/kg 6 < 5 < 10 < 10 SW µg/l 23 56 12 < 10 HW µg/l 17 35 <10 < 10 WTR µg/l 21 39 < 10 < 10 GB µg/l 27 52 14 12 B µg/l 26 51 18 < 10 F o r P e e r R e v i e w Sample Series ART DON-3-Glc ADONs HT-2 14 140 11 <5 11 <10 8 12 <10 13 18 <10 56 146 12 106 148 24 < 5 1 712 349 933 < 10 77 < 5 103 838 50 < 10 59 < 5 116 499 65 < 10 < 5 < 5 219 561 131 < 10 < 5 < 5 237 401 104 11 < 5 < 5 180 267 115 10 < 5 < 5 178 265 117 < 10 7 * NIV,	O n l y	Formatted: Font: 8 pt Formatted: Superscript Formatted: Font: 8 pt Formatted: Font: 8 pt Formatted: Font: 8 pt
	GM	49	68	44	24	601	90	699	20
	KI	46	17	22	37	575	82	314	47

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  Figures 2 and 3 Transfer of DON, DON-3-Glc and ADONs from malt grist to beer (total amount of mycotoxins in malt grist taken for respective beer batch production = 100%; contents of these mycotoxins in analysed samples are shown in TableIX; sample codes are specified in TableVI)

	Sample Series NAT
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

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