ARTICLE pubs.acs.org/JAFC
Deoxynivalenol, Deoxynivalenol-3-glucoside, and Enniatins: The Major Mycotoxins Found in Cereal-Based Products on the Czech Market Alexandra Malachova, Zbynek Dzuman, Zdenka Veprikova, Marta Vaclavikova, Milena Zachariasova, and Jana Hajslova* Department of Food Chemistry and Analysis, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic ABSTRACT: Fusarium toxins, Alternaria toxins, and ergot alkaloids represent common groups of mycotoxins that can be found in cereals grown under temperate climatic conditions. Because most of them are chemically and thermally stable, these toxic fungal secondary metabolites might be transferred from grains into the final products. To get information on the commensurate contamination of various cereal-based products collected from the Czech retail market in 2010, the occurrence of “traditional” mycotoxins such as groups of A and B trichothecenes and zearalenone, less routinely determined Alternaria toxins (alternariol, alternariol monomethyl ether and altenuene), ergot alkaloids (ergosine, ergocryptine, ergocristine, and ergocornine) and “emerging” mycotoxins (enniatins A, A1, B, and B1 and beauvericin) were monitored. In a total 116 samples derived from white flour and mixed flour, breakfast cereals, snacks, and flour, only trichothecenes A and B and enniatins were found. Deoxynivalenol was detected in 75% of samples with concentrations ranging from 13 to 594 μg/kg, but its masked form, deoxynivalenol-3-β-D-glucoside, has an even higher incidence of 80% of samples, and concentrations ranging between 5 and 72 μg/kg were detected. Nivalenol was found only in three samples at levels of 30 μg/kg. For enniatins, all of the samples investigated were contaminated with at least one of four target enniatins. Enniatin A was detected in 97% of samples (concentration range of 20 2532 μg/kg) followed by enniatin B with an incidence in 91% of the samples (concentration range of 13 941 μg/kg) and enniatin B1 with an incidence of 80% in the samples tested (concentration range of 8 785 μg/kg). Enniatin A1 was found only in 44% of samples at levels ranging between 8 and 851 μg/kg. KEYWORDS: ultrahigh-performance liquid chromatography, orbitrap mass spectrometry, bakery products, QuEChERS, natural toxins
’ INTRODUCTION Cereal-based products represent one of the most important dietary items in many countries around the world. Unfortunately, cereal grains, like many other agricultural commodities, can be contaminated by toxic secondary metabolites of various genera of fungi, which may colonize the various substrates at all stages of the production chain. As these secondary metabolites are relatively stable under the common conditions employed in food processing (including heat treatment), these hazardous natural toxins can be transferred into the final products.1 3 For the contamination of cereals occurring in the field, mycotoxins produced by microscopic filamentous fungi of the Fusarium genus are very common in temperate regions.4 On the basis of available occurrence and toxicological data on mycotoxins derived from Fusarium (generally in the field) and also Aspergillus and Penicillium (generally in storage) species, legislation on the maximum permitted limits in many raw and processed products has been established by the European Commission (EC 1881/2006). These limits have been set for deoxynivalenol, the key representative of group B trichothecenes, the sum of fumonisins B1 and B2, zearalenone, aflatoxins, ochratoxin A, and patulin.5 For two other Fusarium mycotoxins, group A trichothecenes HT-2 and T-2 toxins, legislative limits are currently in preparation.5 Changing climatic conditions together with ongoing innovation of agricultural practices have resulted in some changes in the spectrum of Fusarium species invading crops in the field. r 2011 American Chemical Society
Consequently, the extent of mycotoxin contamination and the type of mycotoxins formed are changing continuously.6 In addition to the “traditional” (regulated) Fusarium mycotoxins, other so-called “emerging mycotoxins”, such as enniatins and beauvericin, have been reported to occur in cereals from central and southern Europe, which in the past were rarely monitored.11 These recent reports have clearly demonstrated that Fusarium strains producing these toxic cyclic hexadepsipeptides are not confined to northern Europe.7 9,11 In addition to Fusarium pathogens, cereal crops can be colonized to some extent by other toxinogenic species belonging to the different genera of microscopic filamentous fungi. For example, some strains of Alternaria genus produce toxins such as alternariol, alternariol monomethyl ether, altenuene, and tenuazonic acid;12 Claviceps fungi, common pathogens of rye, are responsible for the production of ergot alkaloids.12 However, reliable occurrence and toxicological data on these other nonroutinely determined mycotoxins are essential for a comprehensive health risk assessment and are still required. With a view to improving this situation, the European Food Safety Authority (EFSA) has issued a call appealing for the collection of occurrence Received: August 23, 2011 Revised: November 9, 2011 Accepted: November 10, 2011 Published: November 10, 2011 12990
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Journal of Agricultural and Food Chemistry data for some of these other toxins in various food- and feedstuffs.13 Another “emerging” food safety issue related to mycotoxins is their “masked” forms. These conjugated mycotoxins have been discovered only relatively recently. Obtaining more knowledge concerning their natural occurrence and particularly the impact of processing/manufacturing practices on toxin levels in the final products has become increasingly important for assessing the potential health risks associated with mycotoxin contamination.14 Masked mycotoxins are formed by living plants during their protective detoxification processes and include the conjunction of mycotoxins to polar substances such as sugars, amino acids, and sulfate and their incorporation into plant cell compartments. Although there was speculation about the existence of masked mycotoxins in the mid-1980s, masked mycotoxins still escape routine analysis for several reasons. As their polarity is higher in comparison to that of precursor toxins, they are hard to extract with the usual solvents and are often lost in the cleanup process; analytical standards were not available, and therefore these masked toxins were not detected.15 Since 2007, when a pure standard of deoxynivalenol-3-β-D-glucoside was introduced on the market, its co-occurrence with deoxynivalenol in wheat, maize,16 and barley17 and after processing has been documented. For example, significant increases of deoxynivalenol-3-β-D-glucoside levels during beer production18 and baking when bakery hydrolytic enzyme-based improvers were added to used flour19 were observed. This might be due to its release from intra-/ intercellular pool or conjugation to glucoside residues during fermentation. Up to now, numerous monitoring studies on mycotoxin contamination in various cereal-based products have been published. However, most of them have focused on the analysis of only one group of related mycotoxins and/or regulated toxins. For example, the occurrence of deoxynivalenol together with some other trichothecenes is usually determined in cereal-based products.20,21 For maize-containing foodstuffs, these have been mainly examined for fumonisins, aflatoxins, and zearalenone.22,23 As a result, there is still a dearth of comprehensive studies in which a wide range of unrelated mycotoxins and their conjugates have been monitored at the same time. The aim of the current study was to fill this information gap by bringing extensive knowledge about mycotoxin contamination of cereal-based products collected from various markets in the Czech Republic in 2010. Attention was particularly focused on the detection of four groups of mycotoxins: (i) trichothecenes and zearalenone; (ii) enniatins and beauvericin; (iii) ergot alkaloids; and (iv) Alternaria toxins.
’ MATERIALS AND METHODS Reagents and Chemicals. Analytical standards of mycotoxins were purchased from three suppliers. Biopure Referenzsubstanzen GmbH (Tulln, Austria) supplied deoxynivalenol, nivalenol, fusarenon-X, 3- and 15-acetyldeoxynivalenol, HT-2 toxin, T-2 toxin, verrucarol, neosolaniol, diacetoxyscirpenol, and zearalenone as pure crystalline substances; deoxynivalenol-3-β-D-glucoside and isotopically labeled 13C15-deoxynivalenol as acetonitrile solution; and ergot alkaloids, namely, ergocornine, ergocristine, ergocryptine, and ergosine, as dry films in vials dedicated for the preparation of solutions. Alexis Biochemicals (New York) provided enniatins A, A1, B, and B1 as pure crystalline standards. Sigma-Aldrich Chemie GmbH
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(Taufkirchen, Germany) supplied pure crystalline standards of alternariol, alternariol monomethyl ether, and altenuene. Stock standard solutions were prepared in acetonitrile and stored at 20 °C. A composite working standard solution in acetonitrile (2.5 μg/mL of 15- and 3-acetyldeoxynivalenol; 5 μg/mL of all other mycotoxins) used for spiking experiments and for calibration purposes was prepared by combining suitable aliquots of each individual standard stock solution. Organic solvents used for extraction and LC-MS analysis as well as ammonium acetate and sodium chloride were obtained from SigmaAldrich. Ultrapure water was produced by a Milli-QSP Reagent water system (Millipore Corp., Bedford, MA). Anhydrous magnesium sulfate and sodium chloride were obtained from Penta (Prague, Czech Republic). Samples. In total, 116 commercially available cereal-based products were purchased randomly from the four biggest international markets in the Czech Republic in 2010 over a 3 month period. The following commodities, listed according to processing method and/or cereal source, were collected. Group 1: White Flour Products, n = 17. These samples included white bread, white bread toasts, and miscellaneous shaped rolls produced only from white wheat flour. Group 2: Mixed Flour and Whole-Corn Products, n = 36. All samples were based on mixtures of wheat and rye flour with the addition of other ingredients such as barley, buckwheat, and oat flours, malt, bran, sesame seeds, and flaxseeds. This sample group was subdivided according to the cost of the products with subgroup 2a being the “budget class”, named whole-corn products, and subgroup 2b being the “superior class”, branded as vital or fit. Group 3: Breakfast Cereals, n = 7. These products included samples of oat, wheat, buckwheat, and barley flakes intended for the preparation of mash, muesli, etc. Group 4: Snacks, n = 34. These products included crispy slices of bread prepared by an extrusion process and consisting of various types of grains (corn, wheat, spelt, rye); puffed snacks prepared mainly from rice with or without the addition of various salty or sweet flavouring (cheese, apple cinnamon); and rusks and party snacks with or without flavoring (garlic, cheese, pizza) prepared from wheat flour. Group 5: Flours, n = 22. The flours used included wheat flour from different stages of milling (from short to smooth flour), three samples of wholemeal flour (wheat, rye, spelt), and one sample of rye bread flour. To ensure representative sampling, five pieces (groups 1 and 2) and five packages (groups 3 5) of each sample were collected and prepared for grinding. Homogenization and grinding were employed using a laboratory knife mill Grindomix GM200 (Vitrum Ltd., Prague, Czech Republic) to obtain a particle size of 1 mm. If necessary, samples from groups 1 and 2 were dried at 40 °C prior to grinding. Weight losses resulting from drying were recorded, and the mycotoxin contents were back calculated so their concentrations were based on the original undried material. Sample Preparation. QuEChERS-Based Method. QuEChERS was introduced for multiple analysis of pesticides in fruits and vegetables.24 This very flexible approach has been adapted with many modifications for the analyses of a wide range of substances including mycotoxins.25,26 The QuEChERS approach previously used in this laboratory for the isolation of analytes from cereals26 was slightly modified (the acidification of water was not necessary for the target analytes involved in this study) and revalidated for bread. In brief, 4 g of homogeneous and representative samples was weighed into PTFE cuvettes, and 7.5 mL of deionized water and 10 mL of acetonitrile were added. The suspension was shaken vigorously by hand for 3 min. After the addition of 1 g of NaCl and 4 g of MgSO4, the mixture was shaken by hand for 3 min again. Prior to the next step, an aliquot of 13C15-labeled deoxynivalenol standard solution corresponding to a contamination 12991
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Table 1. Performance Characteristics of QuEChERS-Based Method Employing LC Orbitrap MS Detection/ Quantitation 100 μg/kg
250 μg/kg a
LCL
recovery
RSD
recovery
RSDa
(μg/kg)
(%)
(%)
(%)
(%)
nivalenol
25
86
13.6
89
9.4
deoxynivalenol
12.5
70
14.3
80
13.1
deoxynivalenol-3-β-D-
12.5
53
7.1
58
9.9
glucoside sum of 3- and 15-acetyl- 12.5
76
11.8
95
9.5
deoxynivalenol fusarenon-X
77
3.3
81
10.8
diacetoxyscirpenol
6.25
91
2.0
95
10.5
neosolaniol
6.25
98
3.0
95
9.8
120
13.9
106
3.3
verrucarol
12.5
62.5
HT-2 toxin
6.25
98
2.9
94
11.1
T-2 toxin zearalenone
6.25 6.25
92 90
3.2 2.4
91 89
13.1 10.3
enniatin A
6.25
86
12.3
80
15.0
enniatin A1
6.25
96
10.8
85
17.4
enniatin B
6.25
94
6.4
82
10.0
enniatin B1
6.25
97
5.9
77
5.0
ergosine
25
72
4.3
78
10.3
ergocryptine
25
57
5.1
72
11.6
ergocristine ergocornine
25 12.5
71 71
6.7 7.1
79 78
9.8 10.9
alternariol
6.25
90
9.7
95
4.7
alternariol
6.25
115
11.3
99
1.6
6.25
107
10.9
99
7.1
monomethyl ether altenuene a
n = 5.
level of 100 μg/kg was added into each sample to control potential losses during partition between an organic and an aqueous phase. The separation of the aqueous phase from organic matter was achieved using centrifugation (5 min, 5000 rpm). In the next step, a 1 mL aliquot of supernatant was transferred into a vial and injected. The performance characteristics of the revalidated method, namely, lowest calibration levels (LCLs), recoveries, and repeatability (relative standard deviations, RSD), obtained for bread are summarized in Table 1. Although possible matrix effects in this survey were not studied in detail within the validation process, corresponding blank matrix samples for each material examined (according to groups 1 5) were used for the preparation of the matrix-matched standards. The linear range of the method used was tested for the matrix-matched calibration curves constructed ranging from LCLs to 500 ng/mL corresponding to 1250 μg/kg of matrix. The majority of analytes showed linearity in the range of 0.9957 0.9999 (R2). “Classic” Method. Previous experience in this laboratory showed that low levels of deoxynivalenol-3-β-D-glucoside in bakery products were expected. As its detection limit expressed as the LCL was only 12.5 μg/ kg and recovery only 53% when using the QuEChERS-based method, it was considered not sufficient for the quantitation of this masked mycotoxin. On this account, for the reliable determination of deoxynivalenol-3-β-D-glucoside it was decided to employ another more timeconsuming analytical approach based on the preconcentration of analytes before injection.19 In brief, 6.25 g of a homogeneous and
representative sample was extracted with 25 mL of an acetonitrile water mixture (84:16, v/v) for 60 min using an automatic shaker (IKA Laboratortechnik, Germany). An aliquot of crude extract (4 mL) was evaporated to dryness, redissolved in 1 mL of a methanol water mixture (1:1, v/v), and subsequently passed through a 0.2 μm microfilter prior to LC-MS analysis. The LCL for deoxynivalenol-3-β-D-glucoside obtained via the classic method developed in this laboratory was 5 μg/kg. Instrumental Parameters. An Accela ultrahigh-performance liquid chromatograph (Thermo Fisher Scientific, San Jose, CA) coupled to an orbitrap mass spectrometer Exactive (Thermo Fisher Scientific, Bremen, Germany) was used for mycotoxin analysis. For the chromatographic separation, a Hypersil GOLD Q column, 100 mm 2.1 mm i.d., 1.9 μm (Thermo Fisher Scientific), held at 40 °C, was used. To achieve the best separation of analytes with narrow peaks, two chromatographic runs had to be employed, and in both cases ammonium formate (5 mM) in water (eluent A) and methanol (eluent B) were used as a mobile phase for both gradients: (i) Gradient I (QuEChERS-based method) started with 10% B and was linearly increased to 25% B in 1 min, followed with another linear increase to 50% B over the next 4 min, then a linear increase of B to 100% over the next 4 min, and a hold at that level for 14 min before a switch back to 10% B. Column equilibration was allowed to take place over the next 4 min before the start of the next run, for which a partial loop injection technique was used. The flow rate was 450 μL/min, and the injection volume was low, at only 2 μL, to avoid undesirable peak distortion. (ii) Gradient II (classic method) started with 5% B and was linearly increased to 50% B in 6 min; over the next 4 min there was another linear increase of B to 100%, which was held at 100% B for 5 min before a switch back to 5% B and then column equilibrium, which was achieved in 3 min. The flow rate was 450 μL/min, and the injection volume was 5 μL, employing a partial loop injection technique. Identification and quantitation of target analytes were performed using single-stage orbitrap mass spectrometry with atmospheric pressure chemical ionization (APCI) (Thermo Fischer Scientific, Bremen, Germany). Optimized operating parameter settings were used: sheath/ auxiliary gas, 55/10 arbitrary units; capillary temperature, 250 °C; vaporizer temperature, 320 °C; capillary voltage, 50/+60 V; discharge current, 5 μA. The system was operated in the full spectrum acquisition mode in the mass range m/z 100 1000 at a resolving power setting of 50000 fwhm with the fixed scan rate of 2 spectra/s. Switching between negative and positive ionization modes was set for the entire run. External mass axis calibration without the use of a specific lock mass was employed, and for the mass accuracy estimation, mass at the apex of chromatographic peak obtained as the extracted ion chromatogram was used. The calculated exact masses of analyte ions are summarized in Table 2.
’ RESULTS AND DISCUSSION To enable the broadest possible range of mycotoxins, a multianalyte method employing ultrahigh-performance liquid chromatography coupled with orbitrap high-resolution mass spectrometry was used for monitoring cereal-based products. Because no Alternaria toxins or ergot alkaloids were detected at levels exceeding their respective LCLs, only results concerning trichothecenes and enniatins are presented. Trichothecenes. The occurrence of trichothecene mycotoxins in five different cereal-based product groups characterized according to the type of ingredients and processing technology used for their production is summarized in Table 3. As expected, deoxynivalenol (Figure 1) was detected in most of the examined samples; its highest incidence rate (94%) was observed in bakery products made from white flour followed by 89 and 73% in mixed-flour products and flour, respectively. There was also a 12992
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Table 2. Overview of Quantitation and Confirmation Ions Together with Relative Intensity Obtained for Particular Mycotoxins by Orbitrap MS Instrument exact massa m/z
analyte nivalenol
retention time
elemental
(min)
formula
[M + H]+
[M + NH4]+
C15H20O7
313.1282
330.1547 (0.08) 314.1598
1.1
[M +Na]+
H]
[M + HCOO]
[M + Cl]
311.1136
357.1191
347.0903
(0.03) 295.1187
(1) 341.1242
(0.02) 331.0953
deoxynivalenol
2.7
C15H20O6
(0.12) 297.1333 (0.32)
(0.13)
(0.05)
(1)
(0.02)
deoxynivalenol-3-β-D-glucoside
2.90b
C21H30O11
459.1561
476.2126
457.1715
503.1770
493.1482
(0.03)
(0.22)
sum of 3- and
3.76
C17H22O7
339.1438
356.1704
(1)
(0.05)
(0.05)
(0.05)
(0.42)
(0.02)
fusarenon-X
2.72
C17H22O8
355.1387
372.1653
377.1207
353.1242
399.1297
413.1453
(0.02) 389.1571
(0.05)
C19H26O7
(1) 384.2017
(0.75)
5.98
(0.66) 367.1751
(0.05)
diacetoxyscirpenol neosolaniol
3.06
C19H26O8
verrucarol
3.33
C15H22O4
15-acetyldeoxynivalenol
(0.03)
(1) 400.1966 (1)
267.1591
284.1856
(1)
(0.83)
361.1258
(0.02)
(1)
(0.02)
337.1293
383.1348
373.1060
(0.03) 405.1520 (0.02) 311.1500 (0.11)
HT-2 toxin
7.08
C22H32O8
425.2170
442.2435
447.1989
T-2 toxin
7.57
C24H34O9
(0.16) 467.2276
(1) 484.2541
(0.02) 489.2095
(0.01)
(1)
zearalenone
7.89
C18H22O5
319.1540
(0.01) 317.1394
(0.21)
(1)
enniatin A
9.58
C36H63N3O9
682.4637
enniatin A1
9.46
C36H61N3O9
enniatin B
9.19
C33H57N3O9
(0.39) 640.4168
(1) 657.4433
622.3987
638.4022
(0.33)
(1)
(0.02)
(0.04)
654.4324
671.4589
676.4143
652.4179
(0.36)
(1)
(0.02)
enniatin B1 ergosine
9.34 7.64
C34H59N3O9 C30H37N5O5
699.4903
704.4456
(0.33)
(1)
(0.03)
668.4481
685.4746
680.4492 (0.06)
684.4077 (0.002)
(0.04)
548.2867
546.2722
(0.95)
(1)
592.2777 (0.02)
ergocryptine
8.17
C32H41N5O5
576.3180
574.3035
620.3090
8.17
C35H39N5O5
(0.78) 610.3024
(1) 608.2878
(0.003)
ergocristine ergocornine
7.90
C31H39N5O5
562.3024
560.2878
606.2933
596.2645
(0.82)
(1)
(0.004)
(0.01)
257.045
(0.61)
(1)
alternariol
6.87
C14H10O5
259.0601
alternariol monomethyl ether
8.20
C15H12O5
273.0758
altenuene
5.28
C15H16O6
(0.45) 293.102
310.1285
(1) 291.0874
(1)
(0.07)
(0.34)
(0.06)
a
[M
(1) 271.0607 337.09289 (0.26)
Quantitation ions are in bold; relative intensities are in parentheses. b Retention time is stated for the classic method.
high incidence of the masked analogue of deoxynivalenol, deoxynivalenol-3-β-D-glucoside, at detectable levels (Figure 1). As shown in a recent study focusing on the impact of milling and breadmaking processes on deoxynivalenol and also
deoxynivalenol-3-β-D-glucoside levels,19 no significant changes of these two toxins took place during kneading and dough fermentation. Only small decreases in deoxynivalenol and its masked form (less than 10 and 20%, respectively) were observed 12993
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Table 3. Fusarium Toxins in Various Cereal-Based Products Involved in the Current Study sample group
no. of positive samples
(no. of samples)
mycotoxin
(% of positive samples)
mean
median
range
white-flour products
deoxynivalenol
16 (94)
125
96
13 350
(17)
deoxynivalenol-3-β-D-glucoside
14 (82)
15
10
5 30
nivalenol
0
enniatin A
16 (94)
112
77
23 325
enniatin A1
11 (65)
17
11
6 39
enniatin B
16 (94)
40
40
15 89
enniatin B1
13 (76)
20
15
9 82
mixed-flour products
deoxynivalenol
32 (89)
139
68
13 431
(36)
deoxynivalenol-3-β-D-glucoside
28 (78)
19
14
7 41
nivalenol
1 (3)
enniatin A
35 (97)
230
80
31 1493
enniatin A1
14 (39)
77
18
6 851
enniatin B
35 (97)
62
52
22 227
enniatin B1
33 (91)
33
24
8 115
breakfast cereals
deoxynivalenol
2 (28)
189
LCL
(7)
deoxynivalenol-3-β-D-glucoside
6 (85)
35
30
19 66
31 347
nivalenol
1 (14)
31
31
31
enniatin A
7 (100)
74
40
36 278
enniatin A1
2 (29)
214
214
18 410
enniatin B
7 (100)
163
35
18 941
enniatin B1
6 (86)
144
17
10 785
snacks
deoxynivalenol
21 (62)
124
36
13 320
(34)
deoxynivalenol-3-β-D-glucoside
28 (82)
32
19
11 94 20 65
nivalenol
1 (3)
enniatin A
34 (100)
41
44
enniatin A1
5 (15)
28
23
24 61
enniatin B
23 (68)
50
34
13 240
enniatin B1
18 (53)
18
17
8 106
flours
deoxynivalenol
16 (73)
103
41
28 594
(22)
deoxynivalenol-3-β-D-glucoside
15 (68)
15
16
5 72
nivalenol
0
enniatin A
21 (96)
300
201
enniatin A1
16 (72)
21
16
7 100
enniatin B
21 (95)
59
41
26 256
enniatin B1
21 (95)
22
15
7 71
during the baking due to the thermodegradation process of compounds (confirmed by the determination of breakdown products).19 Figure 2 illustrates the distribution patterns of deoxynivalenol and deoxynivalenol-3-β-D-glucoside concentrations in various cereal-based food categories. Interestingly, in breakfast cereals and also in cereal snacks, the incidence of deoxynivalenol-3-β-Dglucoside exceeded even that of deoxynivalenol. Other food products in which only deoxynivalenol-3-β-D-glucoside was present (no deoxynivalenol detected) were two samples of a unique brand of bread containing barley malt and malt sprouts. A higher incidence of deoxynivalenol-3-β-D-glucoside than deoxynivalenol in some types of cereal-based products may depend on the type of food processing technology used. However, this
27 2532
assumption cannot be supported within this study because the data obtained did not allow any generalizations to be made regarding the relationship between deoxynivalenol and deoxynivalenol-3β-D-glucoside levels in raw material and in final products. Considering all of the sample groups investigated (except flours), the highest levels of deoxynivalenol found in this study ranged from 320 to 431 μg/kg, and thus none of the samples exceeded the maximum limit of 500 μg/kg established by the European Commission for bread, small bakery wares, pastries, biscuits, cereal snacks, and breakfast cereals. The highest level (594 μg/kg) detected in flour also complied with the legislative limit of 750 μg/kg set for this product.5,27 Deoxynivalenol levels determined in this survey were in relatively good agreement with earlier published studies in other 12994
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Figure 1. Chemical structures of deoxynivalenol (1), nivalenol (2), deoxynivalenol-3-β-D-glucoside (3), and enniatins (4).
Figure 2. Distribution of deoxynivalenol (A) and deoxynivalenol-3-βD-glucoside (B) in various cereal-based products.
countries. For instance, a similar German survey summarizing contamination levels of cereal-based products involving breads, noodles, breakfast cereals, and baby and infant foods reported that deoxynivalenol was a dominating mycotoxin with an incidence between 52 and 93%, with mean levels ranging from 61 μg/kg in baby and infant foods to 158 μg/kg in noodles.20 Another recently published study, concerned with monitoring of group A and B trichothecenes in various types of conventional
and organic breads collected from German retail markets, documented that deoxynivalenol occurred in 93 of 101 samples at a mean level of 155 μg/kg and a concentration range of 15 690 μg/kg.21 On the basis of the knowledge of ongoing research concerned with origination of masked mycotoxins and their behavior during food processing, the levels of deoxynivalenol-3-β-D-glucoside in the final processed foodstuffs seem to be strongly dependent on two factors: (i) the intensity of detoxification processes in living crops and, as mentioned above,15 (ii) the type of processing technology employed.18 As far as the plant’s self-protecting detoxification process results in building of a big pool of masked mycotoxins, which may be released by enzymatic processes during the food production, then the levels of deoxynivalenol3-β-D-glucoside in the final products could be high and perhaps even higher than in respective raw materials.18 The mean levels of deoxynivalenol-3-β-D-glucoside in the current sample set varied from 15 to 35 μg/kg depending on the sample group. As illustrated in Figure 2B, the highest concentrations exceeding 30 μg/kg were detected in breakfast cereals and snacks. Interestingly, levels of deoxynivalenol-3-β-D-glucoside higher than 40 μg/kg were not determined in any of the samples except the bakery products made from white flour. The highest deoxynivalenol-3-β-D-glucoside concentration (72 μg/kg) was detected in one sample of a cereal snack, namely, crispy whole-grain slices. It is worthy of note and surprising that the frequent presence of deoxynivalenol-3-β-D-glucoside in the human diet has not been considered in either the calculation of the Total Daily Intake (TDI) for deoxynivalenol27 or the maximum limits established for deoxynivalenol in various foodstuffs.5 The current authors consider this to be in need of urgent review. With regard to other B group trichothecenes, only nivalenol was detected as a co-occurring toxin with deoxynivalenol and deoxynivalenol-3-β-D-glucoside in the current sample set, albeit 12995
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Figure 4. Comparison of average concentrations of enniatins within various cereal-based products.
Figure 3. Percentage distribution of enniatins in various cereal-based products (4, 3, 2, and 1 denote number of enniatins).
at a low incidence. As summarized in Table 3, only 3 samples, whole-grain roll, wheat flakes, and crispy maize slices, from a total of 116 contained detectable levels of nivalenol (30 μg/kg). None of the target A group trichothecenes and zearalenone were detected in any of the investigated samples. Enniatins. Up to now, rather limited knowledge is available on the transfer of enniatins across the food production chain. The occurrence of relatively high levels (up to mg/kg) of these “emerging” Fusarium mycotoxins in cereal grains, which have been reported in some studies conducted in Norway,8 Finland,7 Italy,11 and Spain,9,11 has raised concerns on the human health risks associated with their potential presence in the diet. Despite an identified gap, practically no information is available on the contamination of cereal-based products that would enable intake estimations of enniatins to be made. Because the current author’s previous pilot study concerned with malting barley harvested in various localities during the 2009 season showed extensive contamination of cereal grains with enniatins (some exceeding 1 mg/kg), it was not unexpected that these mycotoxins were found in wheat grains and products in the current study (Table 3). All four enniatins (A, A1, B, and B1) were detected in most of the investigated samples, and all of them contained at least one representative of this group. Figure 3 shows the percentage distribution of enniatins within the respective sample groups. When the various groups of cereal-based products were compared, the highest occurrence of all four enniatins was observed in flour. Almost 73% of this commodity contained all four enniatins and the remainder at least three of them. On the other hand, snacks were found to be a sample group with the lowest occurrence of enniatins. All four enniatins were detected in only 12% of snack samples, and almost 38% of them contained only one of the enniatins. The sum of average levels of individual enniatins (A + A1 + B + B1) for respective sample groups varied between 161 and 595 μg/kg (Figure 4). Although the highest average contamination level of all enniatins was found in breakfast cereals, the maximum content expressed as the sum of enniatins (2650 μg/kg) was detected in a sample of wheat flour. In contrast to that, the lowest concentration of all enniatins was observed in snacks, and the
least contaminated product with the sum of enniatins at 32 μg/kg was the puffed rice slices. When the pattern of enniatins in respective groups of cereal-based products was assessed, enniatin A was dominate in all sample groups, with a frequency ranging between 94 and 100% and concentrations of 20 and 2532 μg/kg for minimum and maximum levels, respectively. Enniatin B contaminated samples with a frequency of 68 100% of all samples, and the concentrations were lower than those of enniatin A. Enniatin B levels ranged between 13 μg/kg detected in spelt slices and 941 μg/kg found in wheat bioflakes. In contrast to enniatins A and B, there was a lower incidence of enniatins B1 and A1. Enniatin B1 was determined with incidences ranging from 53 to 95%, with a minimum measured level of 8 μg/kg in wheat flour and the highest level of 785 μg/kg detected in a sample of wheat bioflakes. The least common representative of enniatins was enniatin A1, with an incidence ranging from 15 to 72% of samples in tested product groups. The broadest concentration range of 6 851 μg/kg was observed in mixed-flour products. As enniatins have become an issue of high concern only recently, the number of published studies concerning their occurrence in cereal-based products is low. Although no similar survey has been undertaken in the countries of central Europe, three monitoring studies, including various processed cereals (except bakery products), have been conducted to date, two of them in Mediterranean countries10,28 and one assessing enniatin contamination in cereal-based products purchased from Italian and Finnish markets.29 By comparison of the pattern of enniatins in the current sample set with data reported in those studies, it seems that the occurrence and level of each enniatin depend on two factors: the origin of the investigated samples and harvest year. The most prevalent enniatin in breakfast and infant cereals originating in Morocco, occuring in only in 30.8% of samples, was enniatin A1.10 Similarly, enniatin A1 was also the most frequent toxin (97.1% of positive samples) in Tunisian processed cereals.28 Interestingly, enniatin A, which was detected with highest incidence in the current sample set, was minor in Mediterranean studies. On the other hand, enniatins B and B1, with an occurrence of 97.0%, were more abundant than other enniatins in various cereal products from the Italian/Finnish study.29 Even concentrations of enniatins in the Mediterranean studies did not correspond to our results. The mean levels of enniatins ranged between 0.6 and 795 mg/kg and between 25.1 and 156.1 mg/kg in Moroccan and Tunisian samples, respectively,10,28 whereas mean values calculated for the Italian/Finnish samples were in range of 3.8 43 μg/kg.29 Due to the high variability of enniatin type and concentrations in the current and previously published 12996
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Journal of Agricultural and Food Chemistry studies, it was not possible to assess which type of products had significantly higher levels of these fungal metabolites. In response to the last EFSA “Call for scientific data on mycotoxins and phytotoxins”,18 the information on enniatin levels in cereal-based products from the Czech market obtained within this study were provided to the EFSA CONTAM panel to contribute to further refining the estimation of consumers' dietary exposure.
’ AUTHOR INFORMATION Corresponding Author
*Phone: +420 220 443 185. Fax: +420 220 443 184. E-mail:
[email protected]. Funding Sources
This research was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic (Projects NPV II 2B08049, NPV II QI111B154, and MSM 6046137305).
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