Distribution of zearalenone in malted barley fractions dependent on Fusarium graminearum growing conditions

Highlights ► Fungal growth conditions and zearalenone levels in malted barley germ, flour and bran. ► Incubation at 30 °C and water activity 0.98 generally reduces zearalenone levels. ► Large drop of zearalenone after 34 days, 20 °C, water activity 0.95, in all fractions. ► Prediction of contamination and manipulation of storage conditions to reduce mycotoxin content. Abstract Zearalenone (ZON) distribution was measured in main fractions of malted barley, dependent on incubation time (17, 26, 34 days), water activity (0.95 and 0.98) and temperature (20 °C and 30 °C). Malted samples were sterilised and inoculated with Fusarium graminearum . ZON levels were higher ( p < 0.01) in bran and germ than flour under almost all growing conditions. Incubation at 30 °C resulted in generally lower ZON levels in germ and bran, regardless of a w and incubation time. After 34 days, ZON levels in flour from samples that were incubated at the higher temperature rose significantly. At 20 °C ZON concentration showed a bell-shaped concentration profile with increasing incubation time in bran and germ, whilst ZON levels in flour increased at a w 0.98 and dropped at the lower a w . The results indicate the importance of storage conditions for ZON levels in commercially relevant grain fractions of malted barley and help predict existing mycotoxin levels or manipulate storage conditions to reduce ZON content. Keywords Malted barley Zearalenone Fusarium graminearum Water activity Temperature Incubation time 1 Introduction Noxious effects of zearalenone (ZON) on humans and animals, primarily reproductive and developmental disturbances ( Zinedine, Soriano, Molto, & Manes, 2007 ), are a great challenge for the food and feed industry. As a secondary metabolite of Fusarium species, ZON is amongst the most frequently detected mycotoxins in cereals. Widespread contamination with major fungal producers of this mycotoxin ( Krstanović, Klapec, Velić, & Milaković, 2005 ) and its presence (contaminating up to 30% of total cereal production) have been reported in Croatia ( Šegvić Klarić, Pepeljnjak, Cvetnić, & Kosalec, 2008 ). Controlled conditions of barley malt production stimulate Fusarium growth and mycotoxin production. Zearalenone and other mycotoxins can, in turn, be transferred to a range of malt products used in human nutrition, including beer, due to ZON solubility in alcohol. Malt by-products (germ and rootlets detached from grain after kilning) are used as animal food and potential risk of mycotoxin exposure has been reported ( Cavaglieri et al., 2009; Wolf-Hall, 2007 ). Although a number of physical, chemical and biological methods have been tested for ZON removal and detoxification ( Zinedine et al., 2007 ), they may prove inappropriate, costly and/or inefficient in decreasing oestrogenicity of contaminated cereal products. Therefore, measures to prevent ZON accumulation are the most effective approach to reduce exposure to the mycotoxin. Mycotoxin contamination is usually greatest in the outer parts of the seed where fungal attack begins ( Murkovic, Gailhofer, Steiner, & Pfannhauser, 1997; Scudamore & Patel, 2009 ), so highest ZON levels in malted barley are expected in bran and germ, and lowest in flour. Several mycotoxins may permeate the endosperm to a certain degree, and ZON seems to be at the lower end of affinity for this fraction ( Lee et al., 1987; Trigo-Stockli, Deyoe, Satumbaga, & Pedersen, 1996 ). There are, however, limited data on whether the abovementioned distribution changes during storage of contaminated cereals ( Martins & Martins, 2002 ). The aim of this work was to determine how variations in incubation time, temperature and water activity affect ZON accumulation in commercially important fractions of malted barley inoculated with Fusarium graminearum . 2 Materials and methods 2.1 Malting, inoculation and incubation Hulled barley samples, obtained from the Agricultural Institute in Osijek, were weighed (250 g) and put into eight malting dishes. Micro-malting was performed according to MEBAK standard procedure ( MEBAK, 1997 ) in a laboratory incubator (ClimaCell, MMM Medcenter Einrichtungen, München, Germany). The kilning of green malt was also performed according to the MEBAK protocol. After drying, malt was transferred into paper bags and kept at room temperature for three days for moisture equilibration. Malt was dispensed (70 g) into Petri dishes ( d = 14 cm), which was subsequently autoclaved. Water activity of malt was adjusted to 0.95 and 0.98 with sterile distilled water and checked using an a w -metre (Rotronic, Bassersdorf, Switzerland). To ensure constant water activity, plastic containers with 100 mL NaCl solution of identical water activity were placed into plastic bags together with the Petri dishes. F. graminearum , zearalenone-producing mould (CBS 110250, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands), was inoculated from PDA agar by taking a 5-mm-diameter agar disc from the margin of the Fusarium colony. The disc was placed agar-down to the centre of each plate which contained prepared malt. The plates were incubated at 20 °C (room temperature) and 30 °C (optimal temperature for Fusarium growth ( Doohan, Brennan, & Cooke, 2003; Hope, Aldred, & Magan, 2005 )) for 17, 26 and 34 days. Inoculation and incubation of malted barley were performed in duplicate. 2.2 Sample preparation Immediately after incubation, malt was dried for 1 h at 50 °C, followed by degermination, milling in a malt mill (Newman Industries, Sale Creek, TN) and sieving, to obtain bran and flour fractions for analyses. The determination of sample dry weight was performed using a HR73 halogen moisture analyser (Mettler Toledo, Greifensee, Switzerland) by drying fraction samples to constant mass. Extraction and immunoaffinity clean-up of samples were performed according to the manufacturer’s (Vicam, Milford, MA) publication Alternative HPLC Procedure for Corn (05.0 ppm) ( Vicam, 1998 ). 2.3 Zearalenone analysis by LC–MS/MS Samples were chromatographed by a Perkin–Elmer (Waltham, MA) Series 200 HPLC coupled with Perkin–Elmer autosampler, using a Supelco (Sigma–Aldrich, St. Louis, MO) C18 column (125 × 4.6 mm i.d., 5 μm particle size) with a Supelco C18 (20 × 4 mm, 5 μm) precolumn. Mobile phases used for gradient elution A (water) and B (methanol, LC-grade purchased from J.T. Baker, Phillipsburg, NJ) both containing 0.1% formic acid. The flow rate was 250 μL/min, the injection volume was 20 μL, and the column was thermostated at 45 °C. The detector was an API 2000 triple-quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, CA) coupled with turbo-ion spray source (ESI). Analyst software Version 1.4.2 was used for data acquisition and processing. The interface was operated in negative ionisation mode according to Cavaliere, Foglia, Pastorini, Samperi, and Lagana (2005) . Calibration was performed by the method of standard additions using ZON standard (Biopure, Tulln, Austria) and recovery was estimated by spiking blank samples at 20.06 mg/kg and 1.003 mg/kg. The retention time for ZON was 26.4 min. 2.4 Statistical analysis Results were expressed as mean per dry weight and differences were tested by one-way ANOVA. Post-hoc analyses of sample subgroups were performed using Fisher’s Least Significant Difference (LSD) test. A p value <0.05 was considered statistically significant. All analyses were performed using Statistica 8.0 (StatSoft, Tulsa, OK) and Microsoft Office Excel 2007 (Microsoft, Redmond, WA). 3 Results and discussion The recovery experiments showed no detectible ZON in non-inoculated malted barley, and a mean 86.7% of the mycotoxin was recovered from the samples, which is in accordance with EC regulation 401/2006 ( EC, 2006a ). In accordance with literature data ( Scudamore & Patel, 2009 ), a majority of combinations of F. graminearum growing conditions generated significantly higher ( p < 0.01) ZON concentrations in malted barley bran and germ than in flour. For example, after the samples were incubated for 17 days at a w 0.95, 20 °C, bran contained 107 mg/kg dry weight, germ 106 mg/kg and flour 33.4 mg/kg. By day 34, ZON levels decreased markedly in all fractions (to approximately 2–8 ppm), eliminating significant difference between bran and flour. On the other hand, 34 days at a w 0.98, 20 °C, along with an increase of ZON concentration in both bran and flour, reversed the starting difference between fractions. Whilst there was less ZON in flour than germ after 17 days of incubation (46.0 mg/kg versus 66.3 mg/g), after 34 days germ ZON level (43.3 mg/kg) was lower, compared to the other fractions (bran: 106 mg/kg; flour: 90.3 mg/kg). Similarly, a w 0.95 and 30 °C brought about a considerable increase in flour ZON concentration on day 34, surpassing germ and bran levels several-fold ( Figs. 1–3 ). Water activity 0.98 and 30 °C were the only growing conditions which resulted in a higher ZON level in flour (5.35 mg/kg), compared to the other two fractions after 17 days of incubation (bran: 0.99 mg/kg; germ: 3.15 mg/kg); the initial pattern of differences between fractions persisted till day 34. Generally, lower incubation temperature (20 °C) stimulated ZON production in all fractions of barley grain. The established differences in ZON levels between malted barley samples incubated at different temperatures and the same a w and incubation time were highly significant in most cases. Martins and Martins (2002) reported a similar effect of temperature (22 versus 28 °C, a w 0.97, 1456 days of incubation) on ZON accumulation in corn inoculated with F. graminearum . However, in the present study, after 34 days of incubation at a w 0.95 and 30 °C, flour ZON concentration rose to 66.9 mg/kg, which was significantly higher than the mean determined at the lower temperature (2.13 mg/kg) ( Fig. 1 ). In fact, bran and germ also contained more ZON under this set of growing conditions, although the differences did not reach statistical significance, thereby breaking the pattern of higher ZON levels at 20 °C. To examine the effect of a w on ZON concentration, the values at the two water activity levels were compared at specific temperature and incubation time. Zearalenone concentrations were only higher in bran and germ at a w 0.95 after 17 days of incubation at the lower temperature ( Figs. 2 and 3 ). After 26 days at 20 °C, the levels in the fractions stagnated (flour, Fig. 1 ) or increased significantly, especially in bran from samples kept at a w 0.98 and germ at a w 0.95. The final ZON content was significantly higher at a w 0.98 in all fractions. Over the course of incubation time at 20 °C, a trend of reducing ZON concentration at the lower a w was noticeable in flour and bran ( Figs. 1 and 2 ). Quite the opposite happened at the higher a w level, which was in agreement with previously reported peak ZON concentrations on day 35 of incubation in artificially inoculated corn, a w 0.97, regardless of temperature ( Martins & Martins, 2002 ). The germ accumulated the highest levels of the mycotoxin after 26 days ( a w 0.95: 169 mg/kg; a w 0.98: 121 mg/kg) which had quickly dissipated by day 34 ( Fig. 3 ). This could be indicative of exhaustion of nutrient sources since germ is the kernel site where fungal invasion of stored cereals begins ( Bechtel, Kaleikau, Gaines, & Seitz, 1985 ). The mycotoxin accumulated in the germ region might have eventually been metabolised and/or redistributed to other parts of the kernel, especially endosperm, following mycelial growth ( Fig. 1 : 20 °C, a w 0.98). Alternatively, the sudden drop of ZON levels in flour and bran at 20 °C, a w 0.95 ( Figs. 1 and 2 ) and in germ at 20 °C and both water activities ( Fig. 3 ), incubated for 34 days, might reflect analytical limitations of this study. Namely, if ZON was metabolised into some other form (e.g., α-zearalenol, β-zearalenol, zearalenone and zearalenol glycosides, and zearalenone sulphate ( Vendl, Berthiller, Crews, & Krska, 2009 )), they were not detected by the LC–MS/MS. Additionally, in contrast to the present work, non-sterile malting conditions during manufacture imply involvement of competing microorganisms which would increase metabolism of ZON. Nevertheless, a recent study of a range of cereal-based foods determined only the sulphate conjugate of ZON at rather low levels ( Vendl, Crews, MacDonald, Krska, & Berthiller, 2010 ). Pending studies by this group will investigate the effect of malted barley storage conditions on the fate of various ZON conjugates. The fractions from samples incubated at 30 °C had expectedly increased ZON levels at the lower a w for most compared data points (ZON content at a w 0.95 versus content at a w 0.98 and specific incubation time). The high levels in flour after 34 days of incubation were probably the consequence of extensive fungal penetration of endosperm. The spike in flour ZON levels after such incubation time was characteristic for both combinations of growing conditions, and is easily explicable in view of the abundance of nutrients and the stimulating effect of ZON on α-amylase and β-glucosidase activity ( Fu, Han, Zhao, & Meng, 2000 ). Whether the opposite effect noted at 20 °C and a w 0.95 ( Fig. 1 ) revealed a set of growing conditions conducive to the reduction of ZON accumulation in malted barley or it was metabolised as mentioned above, remains to be seen. This work determined significant effects of combinations of F. graminearum growing conditions on the levels of ZON in malted barley grain fractions. An apparent decrease of ZON levels at a w 0.95, 20 °C on day 34 was evident in all fractions ( Fig. 1–3 ). At 30 °C, both a w 0.95 and 0.98, flour storage longer than 26 days ( a w 0.98: 0.91 mg/kg; Fig. 1 ) led to significant ZON increases. The same can be stated for the bran, although only at a w 0.98 (26 days: 0.66 mg/kg); Fig. 2 ). Zearalenone concentration in the malted barley germ reached the lowest values at a w 0.98, 30 °C after 26 and 34 days of incubation (34 days: 0.94 mg/kg; Fig. 3 ). Although kilned barley malt does not support growth of microorganisms, localised high-moisture zones can occasionally form in stored barley malt, leading to spoilage. The incubation temperatures used in this work are often encountered during malt storage at ambient conditions and, whilst these can accelerate loss of brewing capacity, there have also been reports of decreasing mycotoxin levels in malt produced from barley stored at similar temperatures (24 °C) ( Beattie, Schwarz, Horsley, Barr, & Casper, 1998 ). Additionally, malted barley is best used following a short period (around 20 days) of storage, shown to be associated with an improvement of brewing parameters ( Rennie & Ball, 1979 ). Therefore, prediction of ZON contamination of specific grain fractions on the basis of storage conditions (water activity, temperature, storage time) and/or their manipulation may be applicable here, taking into consideration the final intended use of the fraction. This could prove a cheap alternative to prevent the oestrogenic toxin exceeding regulated or guideline levels for food and feed ( EC, 2006b, 2007 ). Acknowledgement This work was supported by the Croatian Ministry of Science, Education and Sports (grants No. 113-1130473-0334 and 113-1780691-0538 ). References Beattie et al., 1998 S. Beattie P.B. Schwarz R. Horsley J. Barr H.H. Casper The effect of grain storage conditions on the viability of Fusarium and deoxynivalenol production in infested malting barley Journal of Food Protection 61 1998 103 106 Bechtel et al., 1985 D.B. Bechtel L.A. Kaleikau R.L. Gaines L.M. Seitz The effects of Fusarium graminearum infection on wheat kernels Cereal Chemistry 62 1985 191 197 Cavaglieri et al., 2009 L.R. Cavaglieri K.M. Keller C.M. Pereyra M.L. González Pereyra V.A. Alonso F.G. Rojo Fungi and natural incidence of selected mycotoxins in barley rootlets Journal of Stored Products Research 45 2009 147 150 Cavaliere et al., 2005 C. Cavaliere P. Foglia E. Pastorini R. Samperi A. 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