Stability of DON and DON-3-glucoside during baking as affected by the presence of food additives

ABSTRACT The mycotoxin deoxynivalenol (DON) is one of the most common mycotoxins of cereals worldwide, and its occurrence has been widely reported in raw wheat. The free mycotoxin form is not the only route of exposure; modified forms can also be present in cereal products. Deoxynivalenol-3-glucoside (DON-3-glucoside) is a common DON plant conjugate. The mycotoxin concentration could be affected by food processing; here, we studied the stability of DON and DON-3-glucoside during baking of small doughs made from white wheat flour and other ingredients. A range of common food additives and ingredients were added to assess possible interference: ascorbic acid (E300), citric acid (E330), sorbic acid (E200), calcium propionate (E282), lecithin (E322), diacetyltartaric acid esters of fatty acid mono- and diglycerides (E472a), calcium phosphate (E341), disodium diphosphate (E450i), xanthan gum (E415), polydextrose (E1200), sorbitol (E420i), sodium bicarbonate (E500i), wheat gluten and malt flour. The DON content was reduced by 40%, and the DON-3-glucoside concentration increased by >100%, after baking for 20 min at 180°C. This confirmed that DON and DON-3-glucoside concentrations can vary during heating, and DON-3-glucoside could even increase after baking. However, DON and DON-3-glucoside are not affected significantly by the presence of the food additives tested.


Introduction
Deoxynivalenol (DON) is a trichothecene mycotoxin produced by fungi of the Fusarium genus, for example F. culmorum and F. graminearum. Environmental conditions that favour DON production in the field are low temperature and high humidity. Cold and wet weather, which tends to delay harvest, permits continued growth of mould on the crop, thereby increasing the chance of higher concentrations of DON being produced (SCF 1999).
In general, acute exposure of animals to DON can result in decreased feed consumption (anorexia), vomiting (emesis), abdominal pain, dizziness, headache, throat irritation, diarrhoea and blood in stools (Rotter et al. 1996). Longer exposure can cause reduced growth, and adverse effects on the thymus, spleen, heart and liver (Sobrova et al. 2010). Monogastric animals, especially swine, show the greatest sensitivity to DON, while chickens and turkeys, as well as ruminants, appear to have higher tolerance (Rocha et al. 2005). Concerning human safety, Scientific Committee on Food (SCF) established a temporary Tolerable Daily Intake of 1 µg/kg body weight per day.
DON occurs predominantly in grains such as wheat, barley, oats, rye and maize, and less often in rice, sorghum and triticale. Thus, DON is found in cereal-based foods (Cano-Sancho, Valle-Algarra, et al. 2011) and feedstuffs (Döll and Dänicke 2011), and this type of food is considered the major contributor of human intake of DON (Cano-Sancho, Gauchi, et al. 2011). Owing to the high presence of DON in cereals, different maximum levels exist for DON in unprocessed cereals and processed cereals/ cereal-based foods (e.g. 1250 µg/kg for unprocessed common wheat and 500 µg/kg for bread and bakery wares) (EC 2006).
Moreover, wheat grains that contain DON may also contain deoxynivalenol-3-glucoside (DON-3glucoside), a metabolite of DON produced by plants (Berthiller et al. 2009). The reported levels of DON-3-glucoside are various, but the concentration ratio DON-3-glucoside/DON is reported to range from around 10 to 30% (Berthiller et al. 2009;Dall'Asta et al. 2013). Moreover, the presence of DON-3-glucoside in processed cereal products has also been confirmed (Suman et al. 2013). Furthermore, Berthiller et al. (2011) showed that DON-3-glucoside can be hydrolysed to DON by several lactic acid bacteria that may be present in the intestine. Therefore, the FAO/WHO Expert Committee (JECFA) has considered DON-3-glucoside as an additional contributing factor of the total dietary exposure to DON (JECFA 2010).
Processing of cereals at high temperatures may affect the DON and DON-3-glucoside content, but thermal food processing seems to be quite variable and dependent on the processing conditions applied: the temperature, the baking time, the type of mycotoxin, the ingredients and the size of baked product. Some studies have reported a significant decrease in the DON level during baking of bakery products (Valle-Algarra et al. 2009;Numanoglu et al. 2012). In contrast, studies by De Angelis et al. (2013) and Zachariasova et al. (2012) have reported that DON is stable during processing steps involving high temperatures. The contradictory published results are sometimes due to the different sizes of assayed products, which affect the heat transfer and favour temperature gradients inside the products (Vidal et al. 2015). Moreover, the possible release of DON from the flour could be due to the presence of some added enzymes (Vidal, Ambrosio, et al. 2016). Similar to DON, DON-3-glucoside stability is also affected by the different sizes of the product, which has caused variable results in past studies (Suman et al. 2012;Zachariasova et al. 2012;Generotti et al. 2015). However, Vidal et al. (2015) showed that DON-3-glucoside may be released under mild baking conditions of temperature and time (for instance, 140°C for 35 minutes or 200°C for less than 10 minutes) but reduced under harsher baking conditions (i.e. longer periods of time and higher temperatures).

DON and DON-3-glucoside contaminated flour
Wheat flour was purchased from a flour mill in Lleida (Spain) and analysed for natural DON and DON-3-glucoside contamination.
To obtain a higher concentration of DON in the contaminated flour, a strain of Fusarium graminearum (TA 3.234; maintained in the Food Technology Dept. collection at the University of Lleida in Spain) was used. It was previously proven to be a DON producer when cultured on wheat flour. The strain was inoculated and incubated in malt extract agar at 25°C until vigorous sporulation occurred. A spore suspension of the mould was prepared in water and Tween 80 (0.005% v/v). Five millilitres of F. graminearum spore suspension were inoculated in a glass flask containing 250 g of flour and 50 mL of water. In total, 3 kg of flour was inoculated with the strain. The flasks were stored at 25°C for 19 days with periodic shaking. Then, the contents of the flasks were powdered, homogenised and subjected to a DON analysis. The content of DON was 12,500 ± 1235 µg/kg (n = 3). The concentration of DON-3-glucoside (a plant metabolite) did not change.
The raw ingredients were manually mixed, and 20 g aliquots were poured into small paper moulds. The pH of each mix was measured with a pH meter (Crison MicropH 2001, Crison Instruments SA, Alella, Spain). From this point, thermoprobes (Proges Plus, Pluck&Track, Thermo bouton) were always used in the bakery matrixes to register the baking temperature. The probes were placed in the centre of the mould before placing them in the oven at 180°C for 20 min. These conditions were established on the basis of previous experiments and are commonly used conditions in the baking industry. After baking, all samples were lyophilised for 72 h, and the samples were stored at −20°C until analysis.

Preparation of mycotoxin standard solutions
A DON standard (Sigma-Aldrich, Alcobendas, Spain) solution was dissolved in ethanol (Sigma-Aldrich, Alcobendas, Spain) at a concentration of 10.0 µg/mL and stored at 4°C. The concentration in the stock solution was checked by UV spectrometry according to the AOAC Official methods of analysis. Working standards (2.5, 1.0, 0.5, 0.1 and 0.05 μg/mL) were prepared by the appropriate dilution of known volumes of the stock solution with the mobile phase and used to obtain calibration curves in an appropriate chromatographic system. A DON-3-glucoside standard (Sigma-Aldrich, Alcobendas, Spain) was dissolved in acetonitrile (J.T. Baker, Deventer, The Netherlands) at a concentration of 10.0 µg/mL and stored at 4°C in a sealed vial until use. Working standards (1.0, 0.5, 0.1, 0.05 and 0.01 μg/mL) were prepared as for DON, and the calibration curves were generated in a similar fashion.

Mycotoxins extraction, detection and quantification
DON and DON-3-glucoside were extracted from 5 g of a lyophilised ground sample (IKA® A11B basic analytical mill, IKA-Werke GmbH & Co. KG, Germany) with 30 mL of distilled water with magnetic stirring for 10 min. The sample was centrifuged for 8 min at Table 1. List of all additives and additional ingredients tested, with the E identification, concentrations used, pH conditions and the average concentration (µg/kg) ± standard deviation with percentage reduction referred to the control for DON (201 ± 28 µg/kg) and DON-3-glucoside (57 ± 11 µg/kg). Five millilitres of filtered sample was loaded onto a DONPREP® IAC column (R-Biopharm, Rhone Ltd, Glasgow, UK), and the column was washed with 10 mL of distilled water. DON and DON-3-glucoside were eluted by consecutively applying 1.5 mL of HPLC-grade methanol (Sigma-Aldrich, Alcobendas, Spain) (with three backflushing steps) and 1.5 mL of milli-Q water (Millipore, Billerica, MA). Zachariasova et al. (2012) confirmed that DON-3-glucoside had good cross-reactivity with the IAC DONPREP® columns (99-102% recovery for DON and DON-3-glucoside when less than 500 ng of these toxins was loaded). The purified extracts were dried under a nitrogen stream at 40°C. Each dried sample was resuspended in 0.5 mL of the mobile phase (water:acetonitrile: methanol, 92:4:4). DON and DON-3-glucoside were determined in an HPLC Waters 2695® system, with a Waters Spherisorb® 5 µm ODS2, 4.6 × 250 mm analytical column coupled with a Waters 2487 UV/Visible dual λ absorbance detector. The absorption wavelength was set at 220 nm. The HPLC mobile-phase flow rate was 0.6 mL/min, the injection volume was 100 μL, the column temperature was 40°C, and the retention times for DON and DON-3-glucoside were 20 and 23 min, respectively.

Methods performance
The analytical methods for DON and DON-3-glucoside were assessed for linearity, precision and recovery with spiked flour and baked products. The possible interference of additives used in the methods performance was not evaluated. Standard curves were generated by the linear regression of the peak areas against concentration (the r 2 values were 0.98 and 0.97 for DON and DON-3glucoside, respectively). The precision was established by determining the DON and DON-3-glucoside levels in the flour and the DON in the bakery product analogue samples in triplicate at least in fortified samples so the recovery rates could be calculated. The limit of detection (LOD) was considered to be three times the signal of the blank reading, and the limit of quantification (LOQ) was set at three times the LOD. The method performance characteristics for DON and DON-3-glucoside are summarised in Table 2.

Statistics
The results were given on a dry-weight basis, and they were not corrected by recovery values. It is worth mentioning that the mycotoxin concentration was not corrected according to the recipe formulation, because the percentage contribution of the additives/ ingredients was small. Three completely independent repetitions were made for each additive treatment. An ANOVA was used to assess the significance of the sample parameters (additive or ingredient additions) in the observed mycotoxin concentrations as well as in the calculated increase or reduction percentages. Correlation analysis was applied in order to check the relation between pH and mycotoxin variation. The software used for statistical analysis was Microsoft Excel® (Microsoft® Corporation, Redmond, WA) and Statistics 20.0 (IBM SPSS® Statistics 20.0 Inc., Chicago).

Initial levels of DON and DON-3-glucoside in flour
The initial DON and DON-3-glucoside concentrations in the natural flour (n = 5) were 252 ± 30 and 60 ± 15 µg/  Flour  60  180  3  300  123 30  41  3  500  87 9  7  DON-3-glucoside  Flour  14  42  3  3  5   50 250 500 80 ± 9 80 ± 5 67 ± 11 12 6 18 a LOD = limit of detection. b LOQ = limit of quantification. c Mean value ± standard deviation. d RSDr = relative standard deviation. kg, respectively. However, an aliquot of the wheat flour was inoculated with a Fusarium mould to get more DON in the flour, to get higher levels in order to be able to see clear differences in the results. For instance, Stanciu et al. (2017) found an average DON concentration of 190 µg/kg in wheat flour from Romania with a maximum concentration of 1787 µg/kg, while Cano-Sancho, Gauchi et al. (2011) found an average DON concentration of 739 µg/kg. Other studies showed more worrying DON concentrations in cereal-based products; DON concentrations over the maximum set up by the EC have been found (SCOOP 2003;Cano-Sancho, Gauchi, et al. 2011;Vidal et al. 2013).
Unfortunately, the DON-3-glucoside concentration could not be modified, because it is formed by the plant. The presence of DON-3-glucoside in raw cereals is also common. Several studies showed the co-occurrence of the two mycotoxins in raw products, and the concentration ratio DON-3-glucoside/DON is described to range from around 10 to 30% (Berthiller et al. 2009;Dall'Asta et al. 2013) as in our flour (ratio DON-3-glucoside/DON = 24%).
The mycotoxins levels found in flour showed that despite all the effort to control the presence of mycotoxins through the food chain, DON and its modified forms still have a wide incidence in wheat products. Moreover, the high levels of DON in cereals cause cereal-based food to be the main source of exposure to this mycotoxin (Marin et al. 2013).

DON stability
The uncooked food matrix had a DON concentration of 350 ± 43 µg/kg. The DON concentration in the flour was 1108 ± 55 µg/kg, but mixing the ingredients reduced the DON by 68% owing to dilution by the flour. A DON concentration reduction by 42% was found in control samples after baking for 20 min at 180°C with an average DON concentration of 201 ± 28 µg/kg. On the other hand, the added ingredients and additives did not cause a significant change in the DON concentration compared with the control (p > 0.05, ANOVA test).
Owing to the high prevalence of DON in cerealbased products , the stability of DON during cereal processing has been deeply studied, but the results among the published studies are highly variable. Baking caused a reduction in the DON in the baked products by 42%. Mycotoxins are considered highly thermostable compounds. They can be reduced during baking, but many factors affect their final concentration. For this reason, existing studies are contradictory regarding fate DON during baking (Neira et al. 1997;Bergamini et al. 2010;Zachariasova et al. 2012;. Product size is a very important factor. Small products as used in this study allow for rapid heat transmission (Vidal et al. 2015). On the other hand, the temperature inside bread loaves is typically below 100°C, regardless of the baking temperature , which is different from our products, for which higher temperatures were registered (Figure 1). The presence of enzymes could affect the stability of DON, for example, xylanase and α-amylase produced increases of DON after baking , because the enzymes released DON embedded in the wheat matrix.
Additives incorporated into the products did not produce changes (p > 0.05, ANOVA test) in DON concentration compared with the control, and a similar reduction was observed (Table 1). Additives such as sorbitol or ascorbic acid are used by the food industry, for example to improve flavour or to extend the shelf-life of bakery products. Some can change the pH of the food (e.g., sorbic acid or citric acid), and DON is sensitive to pH variations. Some additives caused pH decreases to as low as pH = 5.2 (ascorbic acid). However, the pH variation was not correlated with the reduction percentage. Mishra et al. (2014) showed that the DON concentration declined sharply at pH = 3, which is much lower than the values recorded in this study. Chang et al. (2015) did not find any differences in the final DON concentration during the bread-making process in the presence of Lcysteine hydrochloride (E920). Boyacioglu et al. (1993) examined the effect of potassium bromate (E924) and ascorbic acid (E300), and these additives did not change the DON concentration during the bread-making process compared with breads without additives. On the other hand, they detected reductions of up to 40% owing to the presence of an additive compared with the control when they used sodium bisulfite (E222), L-cysteine (E920) and ammonium phosphate (E342i). However, European legislation does not permit the use of ammonium phosphate (E342). Sodium bisulfite (E222) and L-cysteine (E920) cause the breakage of disulfide bonds, which are covalent bonds between the sulfur atoms of two molecules of the amino acid, cysteine. Therefore, DON could be linked to these covalent bonds in the wheat matrix.
Finally, a reduction in DON levels may be accompanied by the appearance of DON reduction products. Mishra et al. (2014) and Vidal et al. (2015) observed the formation of deepoxy-deoxynivalenol as a degradation product of DON after heating at 125-200°C. Moreover, Bretz et al. (2006) detected other degradation products after heating DON, including norDON A, B and C, but a lack of standards for these products did not allow their quantification in this assay.

DON-3-glucoside stability
The DON-3-glucoside concentration was below the LOD (14 µg/kg) in the uncooked food matrix. Although the initial DON-3-glucoside concentration was 60 µg/kg, dilution using the other ingredients caused a reduction in the DON-3-glucoside concentration. It would be interesting to achieve a larger DON-3-glucoside concentration in the initial wheat flour, but as it is formed by plants, it is not possible to modify its concentration in the flour, without direct spiking. However, we were able to detect DON-3-glucoside in the baked products because it increased during the baking step. Hence, the DON-3-glucoside concentration in the control baked product was 57 µg/kg. Thus, baking produced a DON-3-glucoside increase compared with the DON-3-glucoside concentration in the uncooked mix (<LOD).
DON-3-glucoside is a conjugated DON mycotoxin produced by the plant as a detoxification product in which the hydroxyl group in position C13 of DON is replaced by a glucose unit. DON-3-glucoside in cereal grains has been known for years. Sewald et al. (1992) were the first to detect this DON metabolite in maize plants. Assays show a high percentage of DON-3-glucoside in some crops, and even all the analysed cereal samples (wheat, maize, oat and triticale) from some studies had some DON-3-glucoside (Berthiller et al. 2009;Desmarchelier and Seefelder 2010;Rasmussen et al. 2010;Dall'Asta et al. 2013), showing the high presence of this modified mycotoxin form. Because of its high prevalence in cereals, the stability of DON-3-glucoside during baking has been previously studied.
The observed increase in DON-3-glucoside after baking agrees with previous similar studies in which DON-3-glucoside increased (Vaclavikova et al. 2013;Vidal, Marin, et al. 2014, Vidal et al. 2015; however, some studies have shown a reduction in DON-3glucoside after baking (Kostelanska et al. 2011;Simsek et al. 2012). The presence of enzymes (e.g., xylanase, amylase, cellulase, protease and glucose oxidase) (Vidal, Ambrosio, et al. 2016), product size, temperature and baking time (Vidal et al. 2015) seem to be important factors for the stability of DON-3-glucoside during baking. Therefore, mild baking conditions (for instance, 140°C for 35 minutes or 200°C for less than 10 minutes) can cause increases of DON-3-glucoside at the end of baking, while harsher conditions (i.e., longer baking times and higher temperatures) lead to a reduction in DON-3-glucoside, as Vidal et al. (2015) found in their experiments. Therefore, the increase in DON-3-glucoside in our study was expected, because we used mild baking conditions. This increase in DON-3-glucoside level and DON reduction during baking has been previously demonstrated because the concentration ratio DON-3-glucoside/DON is described to range from around 10 to 30% in raw cereals (Berthiller et al. 2009;Desmarchelier andSeefelder 2010, Rasmussen et al. 2010;Dall'Asta et al. 2013), while this ratio in baked products increases, and even in some cases the DON-3-glucoside concentration is the same as the DON concentration (De Boevre et al. 2013). Thus, the DON-3-glucoside increase and the possible DON reduction during baking cause the ratio to increase. Kostelanska et al. (2011) suggested that for DON-3-glucoside, there is a possible link to the cellular polysaccharides of the matrix in raw products through glycoside bonds. This attachment of DON-3-glucoside to the matrix means that DON-3-glucoside is not detected during analysis of raw products. Food processing breaks the glycoside bond and brings about the detected increase in DON-3-glucoside in the processed products.
More studies that examine the relationship between DON and DON-3-glucoside are necessary to fully understand the interactions between the parent and conjugated mycotoxins in food processing.
Additives did not affect the DON-3-glucoside concentration after baking (Table 1; p > 0.05, ANOVA test). Unfortunately, no other studies on DON-3-glucoside stability in the presence of additives exist.
Finally, the increase in DON-3-glucoside during baking is of some concern. In spite of the fact that in this study the initial DON-3-glucoside concentration is low, and consequently its final concentration in the cooked matrix is not high, the large increase in DON-3-glucoside concentration during baking could be more problematic when the initial DON-3-glucoside concentration is higher. On the other hand, although DON-3-glucoside is far less active as an inhibitor of protein biosynthesis than DON (Poppenberger et al. 2003), DON-3-glucoside is likely to be cleaved in the gastrointestinal tract owing to chemical hydrolases or, more importantly, microbial activity in the intestine. The activity of chemical hydrolases and intestinal microbes has been demonstrated in vivo in swine and in vitro using human intestinal microbiota (Berthiller et al. 2011). For this reason, JECFA and EFSA considered including DON-3-glucoside in the total dietary DON exposure (JECFA 2010).

Conclusion
In conclusion, DON and DON-3-glucoside stability is not affected during baking by the presence of the food additives and the ingredients tested. These additives were tested at the common levels applied in the industry (not necessarily at the maximum allowed concentrations) in different kinds of bakery products, although in this study we tested a standardised baked product. Thus, the tested additives should not be taken into account for the establishment of performance objectives regarding DON and DON-3-glucoside content. However, more studies should be carried out with additives used in the food industry using higher concentrations, closer to the maximum level permitted by EU. Moreover, we confirmed that DON and DON-3glucoside concentrations can vary during heating and that DON-3-glucoside increases after baking.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Ministerio de Ciencia y Tecnología;