ORIGINAL PAPER

Stability of sterigmatocystin during the bread making processand its occurrence in bread from the Latvian market

Aleksandrs Veršilovskis & Vadims Bartkevičs

Received: 13 October 2011 /Revised: 13 January 2012 /Accepted: 16 January 2012 /Published online: 8 February 2012# Society for Mycotoxin Research and Springer 2012

Abstract Sterigmatocystin (STC) is a carcinogenic and muta-genic mycotoxin produced by fungi of many Aspergillus spe-cies. The aim of this research was to test the stability of STCduring the bread making process and to check bread samplesfrom the Latvian market for STC contamination, using a pre-viously developed electrospray positive ionisation (ESI+) liquidchromatography-tandem mass spectrometry (LC-MS/MS)method. Wheat grain naturally contaminated with STC wasused for bread baking. STC was found to be stable during thebread-making process. In the food survey 17% of the analysedbreads were positive for STC, with concentration levels of 2-7 μg kg-1. One out of six rye bread samples, one out of ninerye-wheat bread samples and three out of 14 wheat breadsamples were contaminated with STC. Four out of five con-taminated samples contained whole grains as the main ingre-dient. We conclude that whole grain bread may be a possiblesource of STC, although even STC-positive bread samplesidentified in this study contained quite low toxin levels.

Keywords Sterigmatocystin . Mycotoxin . Stability .

Bread .Aspergillus . LC-MS/MS

Introduction

Sterigmatocystin (STC) is a mycotoxin produced by fungi ofmany Aspergillus spp. (A. versicolor, A. chevalieri, A. ruber,A. amstelodami, A. aureolatus, A. quadrilineatus and A.sydowi) (Atalla et al. 2003). The molecular structure of STCis similar to aflatoxin B1 (AFB1) (Fig. 1). It is a precursor ofAFB1 in the biological transformation (Betina 1989). It is lessacutely toxic than AFB1 in rodents and monkeys, but appearsto be slightly more toxic in zebra fish (EMAN 2011). In mice,the LD50 is in excess of 800 mg kg-1. The 10-day LD50 inWistar rats is 166 mg kg-1 in males and 120 mg kg-1 foradministration in females. The 10-day LD50 for vervetmonkeys is 32 mg kg-1. Chronic symptoms include inductionof hepatomas in rats, pulmonary tumours in mice, renallesions and alterations in the liver and kidneys of AfricanGreen monkeys. Rats fed 5–10 mg kg-1 of STC for 2 yearsshowed a 90% incidence of liver tumours (EMAN 2011).Since STC is a carcinogenic compound that has been shownto affect various animal species (Purchase and Van der Watt1970), it was classified as a 2B carcinogen by the InternationalAgency for Research on Cancer (IARC 1976). There aremany reports about toxicity, carcinogenity, mutagenity andteratogenity of STC (Sweeney and Dobson 1999; Tong-xi etal. 2000). So, the main public health threat arises from thecarcinogenicity of STC rather than its oral toxicity. STC is alsoan important contaminant of building materials and dwellings(Engelhart et al. 2002; Nielsen et al. 1999). Natural occurrencein food and food products has been reported, although only alimited number of surveys have been carried out (Veršilovskisand De Saeger 2010). STC has been found in grains (Scott

A. Veršilovskis (*) :V. BartkevičsInstitute of Food Safety, Animal Health and Environment “BIOR”,Laboratory of Food and Environmental Investigations,Lejupes street 3,1076 Riga, Latviae-mail: aleksandrs.versilovskis@yahoo.com

A. VeršilovskisVeterinary and Agrochemical Research Centre (CODA-CERVA),Operational Direction Chemical Safety of the Food Chain,Unit of Toxins and Natural Compounds,Leuvensesteenweg 17,3080 Tervuren, Belgium

A. VeršilovskisFaculty of Pharmaceutical Sciences, Department of Bio-Analysis,Ghent University,Harelbekestraat 72,9000 Ghent, Belgium

Mycotoxin Res (2012) 28:123–129DOI 10.1007/s12550-012-0124-0

1972; Shannon and Shotwell 1976; Mills and Abramson1986; Rao and George 2000), rapeseeds (Mills and Abramson1986), in soybeans (Shannon and Shotwell 1976), cheese(Francis et al. 1987; Lund et al. 1995; AbdAlla et al. 1996;Scudamore et al. 1997), spices (red pepper, caraway, cuminand marjoram) (ElKady et al. 1995), cocoa beans (Hurst et al.1987), vegetables (Thurm et al. 1979), pistachio nuts(Sommer et al. 1976), coffee beans (Purchase and Pretoriu1973) and in feed (Scudamore et al. 1997; Domagala et al.1997). So far, no country has set regulations for STC, howeversome countries before joining the European Union had rela-tively low maximum levels for STC (e.g. Czech Republic andSlovakia had the level 5 μg kg-1 for rice, vegetables, potatoes,flour, poultry, meat, milk, and 20 μg kg-1for other foods)(Stroka et al. 2004). Soon after STC was recognised as ahighly toxic compound, the California Department of HealthServices set up “no significant risk” intake levels for humans.The resulting concentration was 8 mg kg-1 of body weight perday for a 70 kg adult (EMAN 2011).

Several research studies on different mycotoxins, includingaflatoxins (which are structurally related to STC), showed thatthey are relatively stable during food processing and storage(Chu et al. 1975; Oluwafemi 2004; Bullerman and Bianchini2007), therefore they can be transferred from the raw food tothe food products.

Taking in to account previous mycotoxin stability researchstudies, as well as its presence in grains and having no infor-mation about stability of STC during food processing, the aimof this research was to test stability of STC during breadmaking process and to check some bread samples availablein the local Latvian markets for STC contamination, using apreviously developed electrospray positive ionisation (ESI+)liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (Veršilovskis et al. 2007; 2008).

Materials and methods

Bread making from contaminated grains

Awheat grain sample contaminatedwith STC (83±8μg kg-1),obtained from the previous study (Veršilovskis et al.2008), was homogenised for 1 h using a laboratorymixer. This sample was then split into two portions.The first portion was milled in a laboratory mill andthen was homogenised. From this homogenised sample,12 samples (25 g each) were taken for analysis to checkSTC homogeneity. Uncontaminated grains prepared inthe same way were used as controls.

The second part of contaminated grains was used forwhole-wheat flour preparation. The flour obtained was usedfor whole-grain bread baking, for which the followingdough making recipe was used: flour, 1,000 g; yeast, 22 g;water, 700 g; sucrose, 40 g; sodium chloride, 10 g. Breadbaking technology was the following: dough raising 20 min,after fermentation time 40 min at 35-38°C. Bread bakingtime was 17 min at 200-220°C. Dough and bread sampleweights are shown in Table 1.

STC content in baked bread samples was compared withSTC content in grains, from which the bread was baked. Toenable a comparison between the results obtained for grains

OO

O

OH

O

OCH3

H

H

OO

O

OCH3

OO

H

H

(a) (b)

Fig. 1 Chemical structures of sterigmatocystin (a) and aflatoxin B1 (b)

Table 1 Dough and breadsample weights in six replicatebaking experiments

Replicate number Sample weight, g

Dough sample Hot bread sample Chilled bread sample

1 300 282 277

2 300 280 273

3 300 283 275

4 300 278 271

5 300 286 280

6 300 285 278

Average 300 282 276

Standard deviation (SD) 0.1 3.0 3.3

Relative standard deviation (RSD),% 0.1 1.1 1.2

Total 1,800 1,694 1,654

124 Mycotoxin Res (2012) 28:123–129

and bread, the measured STC content in bread was recalcu-lated to the STC content in flour, considering baking ingre-dients and thermal weight losses, using the followingquotations. The formula in Eq. 1 was used for the recalcu-lation of known cold bread mass obtained from the knowndough mass to bread mass that was obtained from 1,000 gflour.

BM1 ¼ DM1 � BM2

DM2; ð1Þ

where the following terms apply:

BM1 bread mass obtained from 1,000 g of contaminatedflour (grains), g (1,628)

BM2 cold bread mass obtained from known dough mass,g (1,654)

DM1 dough mass obtained from 1,000 g of contaminatedflour (it was calculated from dough making recipe),g (1,772)

DM2 average dough mass used for bread making, g(1,800).

Using the result obtained from Eq. 1 (from 1,000 g offlour or grains we got 1,628 g of bread), we have foundcoefficent for STC content recalculation in bread to STCcontent in grains, applying the next formula, Eq. 2.

K ¼ BM1

FM1; ð2Þ

where the following terms apply:

K recalculation coefficient (1.628)BM1 bread mass obtained from 1,000 g of contaminated

flour, g (1,628)FM1 flour mass from recipe, g (1,000).

The recalculation coefficient is 1.628. This coefficientwas applied for recalculation of obtained STC content inbread to STC content in flour, which can be seen in the nextformula, Eq. 3.

FRSTC ¼ K�BRSTC; ð3Þwhere the following terms apply:

FRSTC STC content in flour recalculated from obtainedSTC content in bread, μg kg-1

K recalculation coefficent, 1.628BRSTC STC content determined in bread, μg kg-1.

Bread samples from local supermarkets

Bread samples (six samples of plain rye bread, nine samples ofmixed rye-wheat bread, and 14 samples of plain wheat bread)were purchased from local supermarkets in Riga. The sampleswere crushed and then homogenised before analysis.

Chemicals and reagents

Methanol (HPLC-grade) and acetonitrile (HPLC-grade) waspurchased from Merck (Darmstadt, Germany). Deionisedwater was purified with Millipore Milli-Q Plus system(Millipore, Molsheim, France). STC standard was purchasedfrom Sigma (St Louis, MO, USA). Argon (AGA, Latvia) wasused as a collision gas in the mass spectrometry.

Preparation of standards

A stock solution of a concentration of 500 μg ml-1 was pre-pared by dissolving 5 mg STC in 10 ml acetonitrile/methanol(50:50, v/v). An aliquot of 500 μl stock solution was evapo-rated to dryness under oxygen-free nitrogen at ambient tem-perature and immediately re-dissolved in acetonitrile (5 ml).

The calibrated stock solution (50 μg ml-1) was used toprepare a standard stock solution of 5 μg ml-1 STC, in aceto-nitrile/water (75:25, v/v). This solution was used to spikesamples for recovery experiments, and to prepare workingSTC standard solutions with concentrations of 0.25 μg ml-1,0.1 μg ml-1, 0.05 μg ml-1 and 0.005 μg ml-1, corresponding toSTC concentrations in sample of 25 μg kg-1, 10 μg kg-1,5 μg kg-1 and 0.5 μg kg-1.

Sample preparation

An 25-g amount of homogenised sample was extracted with16% water in acetonitrile (100 ml) for 30 min using ahorizontal shaker. After filtering through a filter paper,10 ml raw extract was diluted with 20 ml water and purifiedusing Strata X (500 mg) SPE column (Phenomenex,Torrance, CA, USA). Before use, the column was condi-tioned with 6 ml methanol, followed by 6 ml water. Then30 ml diluted extract were passed through the column. Thecolumn was washed first with 40% acetonitrile/water, thenwith 40% methanol/water. The STC was eluted with 4 ml100% acetonitrile. The resulting eluate was evaporated todryness under nitrogen at 60°C and redissolved in 250 μlacetonitrile/water (75/25, v/v). The calibrants were preparedby spiking the blank matrix with the standard solutions andprepared in the same way as the samples.

LC-MS/MS analysis

AWaters Alliance 2695 liquid chromatograph (Waters) wasconnected to a MicroMass Quattro LC triple-quadrupolemass spectrometer (Micromass, Manchester, UK). An ESIprobe in the positive mode was used in the analysis of STC.The mobile phase consisted of 0.01 % formic acid in aceto-nitrile and 0.01% formic acid in water (75:25, v/v) used inisocratic regime. The column used was a Phenomenex Luna(2) C18 (particle size, 5 μm), length 150 mm, internal

Mycotoxin Res (2012) 28:123–129 125

diameter 3.0 mm (Phenomenex, Torrance, CA, USA). Theflow rate was 0.3 ml min-1, column temperature was 30°Cand the injection volume was 25 μl. The parameters of themass spectrometer were optimised using the STC standardsolution of 1 μg ml-1. The best response was recorded withthe following parameters: cone voltage 30 V, capillary volt-age 3.5 kV, extractor 2 V, radio frequency (RF) lens 0.2 V,source temperature 120°C and desolvation temperature 350°C,cone gas flow 63 l h-1, desolvation gas flow 553 l h-1, collisionenergy 30 eV. For the structural identification in select-ed reaction monitoring (SRM) mode, the molecular ion[M + H]+ (m/z 325) was fragmented within the MS toits product-ions (325>310 [M-CH3+H]

+ and 325>281[M-CO2+H]

+) collision energy 30 eV, dwell time 0.2 s. Argonat pressure 3.5 bar was used as a collision gas. A calibrationcurve constructed using external standardisation in matrix.The daughter-ion (m/z 281) was used for the quantificationof STC. The ratio between peaks of STC obtained on twoSRM channels (peak area (325>310)/peak area (325>281))was used for confirmation of analyte. This ratio should be0.69±0.14 for the compound to be confirmed.

Spiking for recovery studies

Samples were artificially contaminated at three differentconcentration levels (0.5 μg kg-1, 5 μg kg-1, 25 μg kg-1).Spiked samples of different bread were prepared by addingSTC standard solution using a digital pipette to 25 g pooledintermediate rye, wheat or rye-wheat bread sample in a250 ml flask, which was left for 1 h at ambient temperaturewith occasional agitation to allow the acetonitrile to evapo-rate. Intermediate bread samples were prepared by mixingand homogenising six to ten blank samples of the samebread type (rye, rye-wheat or wheat). Six replicates at eachconcentration level were prepared from each commodity forrecovery experiments. Recovery results for different breadmatrices are shown in Table 2.

Calibration and linearity

In LC-MS methods the matrix often causes the change ofthe response, because the matrix components disturb theionisation of the analytes (Tang and Kebarle 1993). Becauseof the matrix effect, the calibrants were always prepared inblank matrix.

The method was linear for STC from 0.5 μg kg-1 to25 μg kg-1. A tolerance of ±10% accepted for the separatecalibration points for good linearity. The regression line with-out matrix was y013,656x+212 (R2>0.999) and regression

Table 2 Recovery of STC frombread matrix Bread matrix type Spike level,

μg kg-1Mean result(n06), μg kg-1

Standarddeviation, μg kg-1

Relative standarddeviation, %

Meanrecovery, %

Plain wheat bread 0.5 0.6 0.1 8.3 112.0

5.0 4.8 0.2 4.6 95.8

25.0 25.8 1.6 6.2 103.2

Plain rye bread 0.5 0.4 0.1 6.0 82.3

5.0 4.2 0.2 5.3 84.7

25.0 23.5 1.4 5.9 94.0

Mixed wheat-rye bread 0.5 0.4 0.1 8.3 90.3

5.0 4.7 0.2 4.9 92.6

25.0 24.4 1.3 5.4 97.4

Table 3 Homogeneity of STC in bread and contaminated grainsduring experimental bread baking using a naturally contaminated batchof wheat. Recalculation of the toxin content in grain from the data forbread also demonstrates the obvious stability of STC during the breadbaking process

Replicatenumber

STC contentmeasured inbread sample,μg kg-1

STC contentrecalculated tograins, μg kg-1

STC contentmeasured incontaminatedgrain, μg kg-1

1 52 84 83

2 49 79 88

3 50 81 80

4 46 75 79

5 45 73 76

6 46 75 89

7 39 63 90

8 48 78 65

9 54 88 87

10 49 80 79

11 43 70 94

12 53 86 91

Average 48 78 83

Standarddeviation (SD)

4 7 8

Relative standarddeviation (RSD), %

9 9 10

126 Mycotoxin Res (2012) 28:123–129

line in matrix was y04,477x-176 (R2>0.999). On this basis,the method was suitable for the analysis of STC.

Results and discussion

STC content in bread and contaminated grains

The results obtained for STC in the bread baking experi-ments are shown in Table 3. The mean content of STC inbaked bread was 48 μg kg-1, which is equivalent to78 μg kg-1 in grains. The mean STC content in contaminat-ed grains used for bread baking was determined to be83 μg kg-1. The results of t-test show that statistically therewere no differences between results obtained in contaminat-ed grains and results obtained in bread baked from thesegrains [tcalculated (1.7)<tcritical (1.8), p>0.05]. So, in contrast

to AFB1, which could be reduced by 17-88% (Bullermanand Bianchini 2007), STC was found to be fully stableduring the bread making process. This leads to the assump-tion that most likely other cereal-based food products maybe affected by STC contamination as well.

Food survey: bread samples from the local supermarkets

Table 4 shows that one sample of rye bread, one rye-wheatbread and three samples of wheat bread were contaminatedwith STC. Four of five STC-positive samples were preparedusing whole grains. Overall 17.2% of all samples werecontaminated with STC.

The STC concentrations found in bread were quite lowranging from 2.4 μg kg-1 to 7.1 μg kg-1. They did notexceed the former maximum levels for this toxin as set inthe Czech Republic and in Slovakia (20 μg kg-1). However,

Table 4 STC occurence in dif-ferent bread samples

R plain rye bread, RW mixedrye-wheat bread, W plain wheatbread, n.d. not detected(< 0.5 μg kg-1)

Sample no. Bread sample brand name Bread type Bakery Result, μg kg-1

1 “Hanzas” rudzu maize R Hanzas n.d.

2 Īstā rupjmaize R Lāči n.d.

3 “Saules” tumšā pilngraudu R Iļguciema n.d.

4 “Rīgas” rudzu maize R Iļguciema n.d.

5 “Fazer” pilngraudu rudzu R Druva n.d.

6 Rudzu maize R Sono 2.4

7 “Hanzas” rudzu-kviešu RW Hanzas n.d.

8 Īstā saldskābmaize RW Lāči n.d.

9 “Fit Life” maize RW Lāči n.d.

10 Saldskābā maize RW Iļguciema n.d.

11 “Meistara” rudzu maize RW Druva n.d.

12 “Meistara” kublu saldskābmaize RW Druva n.d.

13 Dinaburga RW Dinella n.d.

14 “Saimnieks” pilngraudu RW Sono 7.1

15 Saldskābmaize RW Sono n.d.

16 Fitness maize W Hanzas n.d.

17 “Hanzas” kviešu maize W Hanzas n.d.

18 “Sendviču” graudu maize ar miežu un rudzu pārslām W Hanzas n.d.

19 “Sendviču” graudu maize ar rudzu pārslām W Hanzas 4.4

20 “Sendviču” graudu maize ar auzu pārslām W Hanzas n.d.

21 Īstā kviešu maize W Lāči n.d.

22 „Saules” graudu maize W Iļguciema 3.2

23 Pilsētas kviešu maize W Iļguciema n.d.

24 “Fazer” karaliskās graudu tostermaizītes W Druva 5.6

25 “Fazer” kviešu tostermaize W Druva n.d.

26 “Spēkotava” kliju maize W Druva n.d.

27 “Zeltene” kviešu maize W Dinella n.d.

28 “Rudens” kviešu maize W Dinella n.d.

29 “Kurzemes” kviešu maize W Sono n.d.

Mycotoxin Res (2012) 28:123–129 127

they exceeded the former level set in these countries for rice,vegetables, potatoes, flour, poultry and meat (5 μg kg-1). So,the interpretation of the analytical results in aspects ofpublic health is difficult. In any case, these findings requiresome attention because if STC is regarded as a carcinogen,the intake of low levels still may result in effects. However,the US Californian “no significant risk” intake level forhumans is about 8 μg kg-1 body weight per day for a 70-kg adult (it is approximately 480 μg per day). Using this as areference, the concentrations as found in ready-to-eat breadin this study, most likely are not a serious threat for con-sumers’ health.

Conclusions

This study presents data that indicate a high stability of STCduring the process of bread baking. The results of ourexperiments show that STC in naturally contaminated grainsis not only heat stable but also survives all technologicalsteps involved in bread making. Therefore, the natural oc-currence of STC in cereals will result in a contamination ofbread, especially in whole-grain types of bread.

This assumption was proved in our survey on theoccurrence of STC in different types of bread from theLatvian market, which demonstrated that this mycotoxinindeed occurs in bread, especially in whole-grain bread.Although the determined STC levels were quite low,bread has to be considered as a potentially relevantsource of STC intake. To avoid STC exposure of theconsumer, a more thorough investigation of the occur-rence in bread and other cereal-based food products inLatvia is clearly necessary.

Acknowledgements The present research was sponsored by the In-stitute of Food Safety, Animal Health and Environment “BIOR”,Laboratory of Food and Environmental Investigations and also by theEuropean Science Foundation, National programme Name: “Support indoctoral and postdoctoral programme realisation and investigation”.Project name: “Support in doctoral and postdoctoral programme investi-gation in engineering, agriculture engineering and forestry science fields”.Contract number: VPD1/ESF/PIAA/04/NP/3.2.3.1./0001/0005/0067.

Conflicts of interest None.

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