Fusarium toxins in wheat flour collected in an area in southwest Germany

International Journal of Food Microbiology 72 (2002) 85 – 89 www.elsevier.com/locate/ijfoodmicro

Fusarium toxins in wheat flour collected in an area in southwest Germany Margit Schollenberger *, Helga Terry Jara, Sybille Suchy, W. Drochner, H.-M. Mu¨ller Institute of Animal Nutrition, Hohenheim University, Emil-Wolff-Str. 10, D70599 Stuttgart, Germany Received 18 December 2000; received in revised form 10 July 2001; accepted 17 July 2001

Abstract A total of 60 samples of wheat flour were collected during the first 6 months of 1999 from mills and food stores in an area in southwest Germany. Samples included whole-grain and two types of white flour with these three groups characterized by a high, medium and low ash content. The contents of deoxynivalenol (DON), nivalenol (NIV), 3- and 15-acetyldeoxynivalenol, HT-2 toxin (HT-2), T-2 toxin (T-2) and fusarenon-X (FUS-X) were determined by gas chromatography/mass spectrometry, and those of zearalenone (ZEA), a- and b-zearalenol (a- and b-ZOL) by high performance liquid chromatography with fluorescence detection. FUS-X, a- and b-ZOL were not detected in any sample. Based on incidence and level, DON was the predominant toxin followed by NIV and ZEA for all three flour types. The overall degree of toxin contamination was lower with decreasing ash content. This suggests a localization of the toxins analyzed primarily in the outer parts of the original wheat kernels. The median DON content was significantly ( P < 0.05) higher for wheat flour originating from wheat of conventional than of organic production. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Trichothecenes; Deoxynivalenol; Zearalenone; Wheat flour; Conventional; Organic; Flour types

1. Introduction During recent decades, the contamination of cereal grains with toxic metabolites of Fusarium species has been increasingly documented (Placinta et al., 1999). A survey of A- and B-type trichothecenes in cerealbased foods marketed in an area in southwest Germany has been reported (Schollenberger et al., 1999). To our knowledge, little published information is available on the natural occurrence of Fusarium * Corresponding author. Tel.: +49-711-459-2407; fax: +49-711459-2421. E-mail address: [email protected] (M. Schollenberger).

toxins in wheat flour. These studies were mostly directed to deoxynivalenol (DON), in part also to nivalenol (NIV) and zearalenone (ZEA) (Tanaka et al., 1985; Trucksess et al., 1996; Pacin et al., 1997; Usleber et al., 2000), but only in one case to type A trichothecenes such as T-2 toxin (T-2), HT-2 toxin (HT-2), T-2 triol and T-2 tetraol (Valente Soares and Furlani, 1996). Results of most of these studies were based on a limited number of samples. The main objective of the present study was to determine trichothecenes of both the A-type (HT-2 and T-2) and B-type (DON, 3- and 15-acetyldeoxynivalenol (3-ADON and 15-ADON), fusarenon-X (FUS-X) and NIV) as well as ZEA and a- and bzearalenone (a- and b-ZOL) in samples of wheat flour

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 6 2 7 - 4

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commercially available in an area in southwest Germany. The occurrence of these toxins in samples defined by a low, medium and high ash content was of interest. Differences in the occurrence of DON, NIV and/or ZEA between fractions of dry milled wheat characterized by different extraction rates, and therefore, different ash contents were indicated by laboratory experiments (Young et al., 1984; Tanaka et al., 1986; Lepschy-von Gleissenthal and Su¨ss, 1996; Trigo-Stockli et al., 1996), as well as by the analysis of commercially available wheat flour (Lepschy-von Gleissenthal and Su¨ss, 1996; Trucksess et al., 1996). A further aim of the present study was to compare the DON content of wheat flour originating from wheat of either conventional or organic origin.

2. Experimental 2.1. Sample materials A total of 60 wheat flour samples of different types characterized by ash content were collected during the first 6 months of 1999 from food stores and mills located in an area within 50 km around the city of Stuttgart, southwest of Germany. Samples were defined to belong to different standards. In Germany, commercially available flours are classified into several standards (called ‘‘types’’) based on the mineral (ash) content corresponding to different extraction rates during milling. The milling companies in Germany use the German standard DIN 10355 (Deutsche Norm 10355) for grading flour, with the ash content being analyzed by the producers according to ICC standard No. 104-1. Group A (n = 28) comprised of white wheat flour belonging to types 405 and 550. Type 405 is defined by an ash content of up to 500 mg 100 g 1 flour. Type 550 is defined by an ash content between 510 and 630 mg 100 g 1 of flour. Flours of these two types were combined into one group because of their similar characteristics. Group B (n = 13) comprised of white wheat flour belonging to type 1050, defined by an ash content between 910 and 1200 mg 100 g 1 of flour. Group C (n = 19) was whole-grain wheat flour. Flours of this grade result from an extraction rate of 100%, and in Germany, usually have an ash content

between 1600 and 2200 mg ash 100 g 1 of flour (Mu¨nzing, 2001). Flour samples were derived from wheat of either conventional (n = 36) or organic (n = 24) origin. All samples were stored at 18 C prior to mycotoxin analysis. 2.2. Mycotoxin analyses Extraction and clean up were carried out as described in detail previously by Schollenberger et al. (1998). In brief, the extraction was performed with a mixture of acetonitrile and water followed by liquid/ liquid extraction with hexane. Clean up was carried out by solid phase extraction using a florisil and a cation exchange cartridge. Derivatization was carried out with trifluoraceticacidanhydride with quantitation by GCMS using a Magnum Ion Trap system in the chemical ionization mode with isobutane as reactant gas. Recovery rates for the trichothecenes in wheat were reported previously (Schollenberger et al., 1998). Detection limits ranged between 20 and 120 pg, corresponding to 2 – 12 mg kg 1 of food, and quantification limits were between 6 and 38 mg kg 1. Toxin contents between the detection and quantification limit were calculated as the average of both limits. Determination of ZEA, a- and b-ZOL was carried out as described previously by Schollenberger et al. (1999). After extraction with a mixture of acetonitrile and water, the sample clean up was carried out using an immunoaffinity column. Identification and quantification of ZEA, a- and b-ZOL were carried out by HPLC with fluorescence detection. The detection limit for ZEA and a-ZOL was 1 mg kg 1 and for b-ZOL, 8 mg kg 1, at a signal to noise ratio of 3:1. Recovery rates in wheat were reported elsewhere (Schollenberger et al., 1999). 2.3. Statistics Normal distribution of toxin contents was tested by using the procedure ‘Proc Univariate Normal’ of the SAS/STAT software (SAS Institute, 1987, Version 6.12). In most cases, toxin contents were not normally distributed. Therefore, a two-sample t-test applied to the ranks was used for the determination of statistical significance of the differences. This is similar to a Wilcoxon rank sum test using the t-approximation for the significant level and was performed by the procedure ‘Proc Npar1way Wilcoxon’ of SAS.

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3. Results 3.1. Occurrence of Fusarium toxins in white and whole-grain wheat flour The occurrence of Fusarium toxins in white and whole-grain wheat flour samples belonging to three groups characterized by different ash contents is presented in Table 1. The predominant toxin was DON for all groups. Based on total samples, the incidence of DON, NIV, 3-ADON, 15-ADON, HT2, T-2 and ZEA amounted to 98%, 12%, 2%, 3%, 7%, 2% and 38%, and the median content of the positive samples amounted to 199, 25, 11, 15, 12, 4, and 3 mg

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kg 1, respectively. FUS-X, a- and b-ZOL were not detected in any of the samples. With regards to the ranking of toxins other than DON, ZEA was found at highest incidence followed by NIV, whereas the median level was highest for NIV. The overall degree of toxin contamination tended to be lower with decreasing ash content (Table 1). Each of the four toxins, 3- and 15-ADON, HT-2 and T-2 was detected in whole-grain flour with the highest ash content (group C), while in white flour containing a medium ash content (group B), only HT-2 was present, and none of the four toxins was found in white flour containing the lowest ash level (group A). Two other toxins, NIV and ZEA, were found in all

Table 1 Fusarium toxins in white and whole-grain wheat flour collected around the city of Stuttgart, southwest Germany * 1

Flour type

Toxin

Number of positive samples (%)

Toxin in positive samples (mg kg Range

Mean

Median

White wheat flour Group A

DON NIV 3-ADON 15-ADON HT-2 T-2 ZEA a-ZOL b-ZOL FUS-X DON NIV 3-ADON 15-ADON HT-2 T-2 ZEA a-ZOL b-ZOL FUS-X DON NIV 3-ADON 15-ADON HT-2 T-2 ZEA a-ZOL b-ZOL FUS-X

28 (100) 1 (4) 0 0 0 0 3 (11) 0 0 0 12 (92) 1 (8) 0 0 1 (8) 0 4 (31) 0 0 0 19 (100) 5 (26) 1 (5) 2 (11) 3 (16) 1 (5) 15 (79) 0 0 0

15 – 965 25

239 F 228 25

156a 25

1–2

1 F 0.6

38 – 756 25

234 F 215 25

12

12

12

1–8

4 F 2.9

4ac

15 – 1379 25 – 40 11 15 12 4 2 – 24

404 F 427 28 F 7 11 15 12 4 6 F 6.67

White wheat flour Group B

Whole-grain wheat flour Group C

)

1a

157a 25

284a 25 11 15 12 4 4bc

* Group A contained 28 samples defined by an ash content of up to 630 mg 100 g 1 of flour. Group B contained 13 samples defined by an ash content between 910 and 1200 mg 100 g 1 of flour. Group C contained 19 samples defined by an ash content between 1600 and 2200 mg 100 g 1 of flour. Values in the final column not sharing a common superscript are significantly different ( P < 0.05) between groups.

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Table 2 Deoxynivalenol in wheat flour produced from cereals of conventional or organic origin * Type

Conventional Organic

Number of samples

36 24

Samples positive (%)

100 96

DON in positive samples (mg kg

1

)

Range

Mean

Median

65 – 1379 15 – 756

394 F 341 131 F 150

295a 120b

* Values in the final column not sharing a common superscript are significantly different ( P < 0.05) between groups.

three groups with decreasing frequency along with decreasing ash content. The level of ZEA tended to decrease with decreasing ash content with a significant ( P < 0.05) difference between the median ZEA content of groups A and C. The incidence of DON was nearly the same for all three groups. However, its content tended to be lower in white compared to whole-grain flour. 3.2. Differences between production methods The total of the samples was divided into two groups according to their origin from wheat of either conventional or organic origin. As demonstrated in Table 2, the incidence of DON was nearly the same for the two groups, whereas the median content was significantly ( P < 0.05) higher in flour samples originating from wheat of conventional production.

4. Discussion The high frequency of DON in wheat flour, with almost 100% of the samples tested, indicates that the original wheat samples were contaminated at least to the same level. This is consistent with the previous findings concerning the occurrence of DON in wheat harvested in the same area in southwestern Germany from which wheat samples of the present survey were collected. In these surveys, the frequency of DON per year ranged between 83% and 96% (Mu¨ller et al., 1997, 2001), which was markedly higher than that of other trichothecenes and of ZEA. Regarding wheat flour, the prevalence of DON compared to other Fusarium toxins was described by Tanaka et al. (1985). These authors examined 36 wheat flour samples for DON and NIV and 27 of these samples also for ZEA. Frequencies of these toxins were 72%, 34% and 11% with average levels in the positive samples

of 38, 24 and 3 mg kg 1, respectively. Pacin et al. (1997) found DON in each of the 61 samples of the wheat flour analyzed. According to Valente Soares and Furlani (1996), neither DON nor NIV, ZEA, T-2, HT-2, T-2 triol, T-2 tetraol and some other mycotoxins were detected in wheat flour, as well as in a variety of other wheat-based products and in whole wheat. This negative result may be attributed to a limited number (between 1 and 11) of the samples analyzed. The results of the present study confirm the view of a localization of Fusarium toxins primarily in the outer parts of grain kernels. Typically, the mineral content is restricted mainly to these parts. Therefore, during the milling process, the ash content increases, related to the whole kernel in fractions produced mainly from pericarp and aleurone tissues, and decreases in fractions produced mainly from the endosperm. The same redistribution was demonstrated in laboratory milling experiments for DON, NIV and ZEA (Tanaka et al., 1986; Lepschy-von Gleissenthal and Su¨ss, 1996; Trigo-Stockli et al., 1996). In these studies, the contents of DON and NIV in wheat flour were reduced by 10 –85% and 20– 70%, respectively, in comparison to the whole-grain, whereas ZEA was reduced by between 30% and more than 90%. A reduction in ergosterol, a chemical indicator for fungal biomass, in white wheat flour compared to the wholegrain was also found (Young et al., 1984). This indicates the localization of fungal mycelium primarily in the outer parts of kernels and is consistent with the result of microscopical analyses which showed a preferred occurrence of Fusarium hyphae in aleurone and pericarp tissues (Bechtel et al., 1985). From these results, it was concluded that DON, NIV and ZEA were localized at the site of their production rather than being transported from the kernel surface to the interior. The present study strongly suggests that not only DON, NIV and ZEA but also 3- and 15-ADON,

M. Schollenberger et al. / International Journal of Food Microbiology 72 (2002) 85–89

T-2 and HT-2 were restricted mainly to the outer parts of the original wheat kernels. The DON content of flour samples originating from conventionally produced wheat was significantly higher than that of samples from organic production (Table 2). Principally, the same ranking of production methods with regards to the content of DON was found previously for cereal-based foods marketed in 1998 in southwest Germany including bread and related products, noodles, breakfast cereals, baby and infant foods, rice and some other cereal-based food products (Schollenberger et al., 1999). Similar results were obtained by Usleber et al. (2000), who found lower DON contents in flour samples of organic origin than in those from conventional production. Somewhat different results were described by Marx et al. (1995). According to a study carried out by these authors in southern Germany, the incidence of DON in conventionally and organically produced wheat was 88% and 76%, whereas the mean contents were 420 and 486 mg kg 1, respectively. No significant difference in DON concentration was observed between the samples of wheat harvested in an area in northwestern Germany from either conventional or integrated production (Berleth et al., 1998). The latter type of production uses conventional methods to fulfill the requirements. Further research is warranted on the question of what is the effect of production methods of the original cereals on the contamination of cerealbased foods with Fusarium toxins. Acknowledgements The technical assistance of L. Burkovski, G. Lai Dinh, S. Planck and M. Ru¨fle is gratefully acknowledged. We thank R. Mosenthin and K. Mu¨nzing for the critical evaluation of the manuscript. References Bechtel, D.B., Kaleikau, L.A., Gaines, R.L., Seitz, L.M., 1985. Effects of Fusarium graminearum infection on kernels from scabby wheat. Cereal Chem. 62, 191 – 197. Berleth, M., Backes, F., Kra¨mer, J., 1998. Schimmelpilzspektrum und Mykotoxine (Deoxynivalenol und Ochratoxin A) in Getreideproben aus o¨kologischem und integriertem Anbau. Agribiol. Res. 51, 369 – 376.

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