Effects of oral exposure to sodium sulphite-treated deoxynivalenol (DON)-contaminated maize on performance and plasma concentrations of toxins and metabolites in piglets

The objective of the present study was to demonstrate the efficiency of the decontamination process applied to deoxynivalenol (DON)-contaminated maize by sodium sulphite (Na₂SO₃) treatment in vivo. Additionally, in vitro characterisation of the toxicity of the DON sulphonates (DONS 1, 2 and 3 denote structurally different forms), the resulting DON metabolites, on peripheral blood mononuclear cells (PBMC) should substantiate the inactivation of DON. In a piglet experiment, both DON-contaminated maize and -uncontaminated control maize either untreated (DON?, CON?) or Na₂SO₃-treated (DON+, CON+) were mixed into feed and fed for 42 d starting from weaning. The results showed that feed intake and daily weight gain of animals fed DON? were significantly lower compared to animals fed CON? and CON+, whereas group DON+ reached the control level or even exceeded it. The feed-to-gain ratio was unaffected (p = 0.45). Furthermore, DON concentrations in plasma markedly reflected the diets’ DON concentrations. These were < 0.1, < 0.1, 5.4 and 0.8 mg/kg feed for CON?, CON+, DON? and DON+, and amounted to 0.3, 0.4, 33.0 and 9.3 ng/ml in plasma, respectively. Whereas DONS 2 and 3 were detected in the DON+ diet, only DONS 2 was recovered in plasma. Regarding the toxicity of DONS, no or much lower toxicity was found compared to DON. DONS 1 and Na₂SO₃ did not affect the viability of PBMC. At 32.71μM DONS2 the viability was reduced by 50% and thus this compound was less toxic than DON by a factor of 73. Consequently, wet preservation of maize with Na₂SO₃ was an effective tool to avoid the adverse effects of DON on performance of piglets.     

Determination of deoxynivalenol-sulfonate (DONS) in cereals by hydrophilic interaction chromatography coupled to tandem mass spectrometry

Deoxynivalenol (DON) is one of most widespread mycotoxins in cereal commodities, and animal feed is prevalently contaminated at high concentrations. This poses a problem in animal nutrition as especially pigs are very sensitive to DON. An effective process for the reduction of the DON concentration is the treatment of contaminated feed with sodium bisulfite (SBS) whereby DON is transformed into DON-sulfonate (DONS). Although the success of this treatment has been confirmed in several feeding studies, it is unexplained if the decrease of DON is accompanied with a coincident increase of DONS. For this reason, we developed a method for the analysis of DONS using hydrophilic interaction chromatography coupled to tandem mass spectrometry. In order to investigate the correlation between DON and DONS concentrations during SBS-treatment, DON-contaminated wheat was treated with SBS and stored for up to 36 days. At defined timepoints of this treatment, samples were analyzed for DON and DONS using stable isotope labeled standards. The preparation, purification, and structure elucidation of DONS, and the HILIC-HPLC-MS/MS method for the analysis of DONS as well as the results of two storage experiments are presented in this paper.     

Investigations on the kinetics of the concentration of deoxynivalenol (DON) and on spoilage by moulds and yeasts of wheat grain preserved with sodium metabisulfite (Na2S2O5, SBS) and propionic acid at various moisture contents

Unground wheat kernels contaminated with 2.09 mg deoxynivalenol (DON) per kg dry matter were stored for up to 56 days at moisture contents of 15, 17.5 and 20% to study the alterations of DON concentration when the wheat was stored either unsupplemented or supplemented with 5 g sodium metabisulfite (Na₂S₂O₅, SBS), 10 g propionic acid (PA) or 5 g SBS plus 10 g PA per kg. SBS addition alone or in combination with PA reduced the DON contamination to 1.2–4.3% of the initial DON concentration while DON concentration of unsupplemented and wheat batches supplemented only with PA varied inconsistently or remained unchanged. The SBS-related DON reduction was paralleled by a concomitant increase in the concentration of the non-toxic reaction product DON sulfonate. In contrast to the unsupplemented wet-stored controls, SBS addition prevented the growth of moulds and yeasts when added alone or in combination with PA. In conclusion, for the conditions examined, the wet preservation of DON-contaminated wheat with SBS seems to be promising as an on-farm detoxification measure.     

Effects of a Fusarium toxin-contaminated triticale, either untreated or treated with sodium metabisulphite (Na2S2O5, SBS), on weaned piglets with a special focus on liver function as determined by the 13C-methacetin breath test

The aim of the present experiment was to test the effects of a wet preservation of triticale contaminated mainly with deoxynivalenol (DON) with sodium metabisulphite (Na2S2O5, SBS) on growth performance, liver function, clinical-chemical plasma parameters and organ histopathology of piglets. For this purpose both the uncontaminated control triticale and the DON contaminated triticale were included in the piglet diet either untreated (CON, FUS) or SBS-treated (CON-SBS, FUS-SBS) and fed for 28 d starting from weaning. The dietary concentrations of DON and DON sulfonate (DONS), the DON derivative resulting from the SBS treatment, amounted to 0.156, 0.084, 2.312 and 0.275 mg DON per kg CON, CON-SBS, FUS and FUS-SBS diet, and to <0.05, <0.05, <0.05 and 1.841 mg/kg diet, respectively. Feeding the FUS diet significantly reduced the feed intake compared to the other three groups as indicated by the significant interactions between triticale source and SBS treatment when the whole experimental period of 28 d was considered (p = 0.014) while live weight gain and feed to gain ratio remained unaffected. The total plasma protein concentration was significantly depressed due to feeding the contaminated diets whereas SBS treatment exerted an increasing effect at the same time (45.4, 49.5, 40.7 and 46.5 g/l for piglets fed the CON, CON-SBS, FUS and FUS-SBS diet, respectively). The liver function was tested by the 13C-methacetin breath test (MBT) allowing evaluation of the cytochrome P4501A2 activity. MBT results, expressed as cumulative percentage dose recovery after 360 min (cPDR360) revealed a slight stimulation of liver function due to SBS treatment (p = 0.052) (37.5, 39.4, 37.4 and 55.1% for piglets fed the CON, CON-SBS, FUS and FUS-SBS diet, respectively). Liver weight and histopathological scoring were only weakly related to the MBT results. Further histopathological examinations of kidneys, pancreas and heart revealed no treatment effects. It was concluded that the SBS treatment of the contaminated triticale restored the performance of piglets to the level of the piglets fed the control diet while the effects on liver function, clinical-chemical plasma parameters - excepting the protein concentration - and organ histopathology were only marginal.     

Occurrence of different trichothecenes and deoxynivalenol-3-β-d-glucoside in naturally and artificially contaminated Danish cereal grains and whole maize plants

Fusarium mycotoxins such as deoxynivalenol (DON) can occur in cereals conjugated to glucose and probably also to other sugars. These conjugates, which are often referred to as ??masked mycotoxins??, will not be detected with routine analytical techniques. Furthermore, it is suspected that the parent toxin may again be released after hydrolysis in the digestive tracts of animals and humans. Today, our knowledge of the occurrence of these compounds in cereal grains is limited. In this paper, a LC-MS/MS method for the simultaneous determination of DON, deoxynivalenol-3-??-d-glucoside (DON-3-glucoside), 3 acetyl-DON, nivalenol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin in naturally (n?=?48) and artificially (n?=?30) contaminated cereal grains (wheat, barley, oat, rye triticale) is reported. The method has also been applied to whole fresh maize plant intended for production of maize silage (n?=?10). The samples were collected from the harvest years 2006?C2010, The results show that DON-3-glucoside and DON co-occurred in cereal grains and, especially in several of the highly contaminated samples, the concentration of the glucoside can be relatively high, corresponding to over 37?% of the DON concentration. The DON-3-glucoside levels in both the naturally and in the artificially grain inoculated with Fusarium were second only to DON, and were generally higher than those of the other tested trichothecenes, which were found at low concentrations in most samples, in many cases even below the detection limit of the method. This argues for the importance of taking DON-3-glucoside into account in the ongoing discussion within the European Community concerning exposure re-evaluations for setting changed values for the tolerable intake for DON. Our results indicate that, in the naturally contaminated grains and in the Fusarium infested cereal grains (winter and spring wheat, oat, triticale), the concentration level of DON-3-glucoside is positively correlated to the DON content. When the DON concentration is high, then the content of DON-3-glucoside will most probably also be high and vice versa.     

Effects of increasing concentrations of sodium metabisulphite (Na<Subscript>2</Subscript>S<Subscript>2</Subscript>O<Subscript>5</Subscript>, SBS) on deoxynivalenol (DON) concentration and microbial spoilage of triticale kernels preserved without and with propionic acid at various moisture contents

Unground triticale kernels contaminated with 6.63 mg deoxynivalenol (DON) per kg dry matter were stored for up to 63 days at total moisture contents of 13 and 15% in order to study the time-dependent kinetics of DON concentration in dependence on graded levels of sodium metabisulfite [0, 1, 2, 3, 4 and 5 g Na₂S₂O₅ (SBS) per kg], and in the absence and presence of 10 g propionic acid (PA) per kg. The DON concentration decreased with increasing amounts of supplemented SBS and with increasing duration of the preservation period in a bi-exponential fashion when SBS addition was ≥3 g/kg. Lower SBS concentrations yielded inconsistent results. The maximum measured DON reductions after adding 5 g SBS/kg were 3 and 4% of the initial DON concentration after 63 days in the absence and presence of PA at moisture contents of 15%, while the corresponding recovery for the variants preserved at 13% amounted to 21 and 11%, respectively. The 12 variants preserved without PA supplementation were more frequently contaminated by moulds and yeasts (n = 5) than the corresponding variants stored together with PA (n = 1). The overall results and regressive evaluations do suggest that the highest SBS addition of 5 g/kg triticale at a moisture content of 15% preserved for 63 days would be necessary for a maximum DON reduction. Although PA did not exert a direct decontaminating effect, an additional supplementation together with SBS seemed to be advantageous with regard to the prevention of yeast and mould contamination and favouring the decontamination reaction by the acid milieu.     

Hydrothermal treatment of naturally contaminated maize in the presence of sodium metabisulfite, methylamine and calcium hydroxide; effects on the concentration of zearalenone and deoxynivalenol

Fusarium toxin-contaminated ground maize was hydrothermally treated in the presence of different combinations of chemicals in order to simultaneously reduce zearalenone (ZEA) and deoxynivalenol (DON) concentrations. Treatments were carried out in a laboratory conditioner at 80 °C and 17 % moisture. Six different treatments were performed, consisting of 3 doses of methylamine (MMA; 2.5, 5 and 10 g/kg maize) at a constant dose of 5 g sodium metabisulfite (SBS)/kg, either with or without the addition of 20 g calcium hydroxide (Ca(OH)₂)/kg. The used maize was contaminated with approximately 45.99 mg DON/kg and 3.46 mg ZEA/kg. Without the addition of Ca(OH)₂, DON reductions reached approximately 82 % after 1-min treatment and the toxin disappeared nearly completely after 10 min when 2.5 or 5 g MMA were applied. ZEA concentrations were only marginally affected. In the presence of Ca(OH)₂, reductions in DON concentrations were lower, but were enhanced by increasing doses of MMA. ZEA concentrations were reduced by 72, 85 and 95 % within the first 5 min of the treatment at MMA dosages of 2.5, 5 and 10 g/kg maize, respectively. The application of SBS in combination with a strong alkaline during hydrothermal treatment seems to be a promising approach to simultaneously decontaminate even high amounts of DON and ZEA in ground maize and may contribute to reduce the toxin load of diets     

Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (k_cat/K_m) of 6.4 mM⁻¹ s⁻¹. The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat.     

Effects of deoxynivalenol and sodium meta-bisulphite on nutrient digestibility in growing pigs

ABSTRACT

Deoxynivalenol (DON), a mycotoxin synthesised by the Fusarium, is known to affect the growth of pigs. This effect can be attenuated with sodium meta-bisulphite (SBS). The aim of this study was to evaluate the effect of SBS with antioxidant blend on nutrient digestibility in pigs fed a diet contaminated naturally with DON. Six crossbred castrated pigs fitted surgically with single-T cannulas in the distal ileum received one of four barley-corn-soybean diets with or without SBS. After 8 d of feeding, faeces and ileal digesta were collected for 2 d. Apparent ileal digestibility (AID) of the dry matter (DM), energy, nutrients and DON, and apparent total tract digestibility (ATTD) of DM, acid detergent fibre (ADF), neutral detergent fibre (NDF), energy and DON were evaluated. The AID of phosphorus, calcium and some amino acids was increased (p < 0.05) in the DON diets whereas the ATTD of DM and energy tended to decrease (p = 0.064 and p = 0.071). SBS reduced the AID of DM, energy, ADF, ether extract, phosphorus and DON (p < 0.05) but had no effect on the ATTD of DM, energy, fibre or DON. These results show that DON improved the AID of some nutrients but tended to reduce the ATTD of energy, which could explain, although anorexia is the main effect of DON on live weight gain, the reported negative effect of DON on pig growth. Finally, SBS with antioxidant blend had reduced AID of some nutrients and intestinal absorption of DON.     

Toxicity,of extracts of barley kernels inoculated withFusarium spp. toArtemia salina

Toxicity toA. salina, of the Fusarium metabolites: deoxynivalenol (DON), its acetylated derivatives (3- and 15-AcDON), zearalenone (ZON), neosolaniol (NEO), nivalenol (NIV), T-2, HT-2 toxins, has been examined and compared with toxicity of extracts of barley kernels (8 cultivars and 4 lines) inoculated withFusarium culmorum, F. graminearum andF. sporotrichioides respectively. Estimated LC₅₀ values were expressed as relative toxicity (RT) in mg DON/kg for samples inoculated withF. culmorum, F. graminearum or in mg T-2/kg forF. sporotrichioides inoculations. Toxicity of extracts of the same genotype/line kernels was compared among different pathogens used for inoculation and differences in Fusarium head blight susceptibility of different genotypes/lines inoculated with the sameFusarium strain were found. Significant correlation between toxicity of extracts (LC₅₀, RT) and toxic metabolites concentration was found ( $\bar r = 0.82$ ; P = 0.01). Bioassays withA. Salina offer a fast, easy and inexpensive method to examine cereal genotypes susceptibility to Fusarium head blight and mycotoxins accumulation in kernels.     

Characterization of a bifunctional wheat inhibitor of endogenous α-amylase and subtilisin

A bifunctional α-amylase/serine protease inhibitor which inhibits germination-specific cereal α-amylases of the Graminae subfamily Festucoideae as well as bacterial subtilisins has been isolated from wheat grains. This protein has M_r ≈20500 and pI ≈7.2. The amino acid composition and N-teminal sequence (45 residues) show that the inhibitor is homologous with cereal and leguminous inhibitors of the soybean trypsin inhibitor (Kunitz) family.     

Prediction of CP and starch concentrations in ruminal in situ studies and ruminal degradation of cereal grains using NIRS

Ruminal in situ incubations are widely used to assess the nutritional value of feedstuffs for ruminants. In in situ methods, feed samples are ruminally incubated in indigestible bags over a predefined timespan and the disappearance of nutrients from the bags is recorded. To describe the degradation of specific nutrients, information on the concentration of feed samples and undegraded feed after in situ incubation (‘bag residues’) is needed. For cereal and pea grains, CP and starch (ST) analyses are of interest. The numerous analyses of residues following ruminal incubation contribute greatly to the substantial investments in labour and money, and faster methods would be beneficial. Therefore, calibrations were developed to estimate CP and ST concentrations in grains and bag residues following in situ incubations by using their near-infrared spectra recorded from 680 to 2500 nm. The samples comprised rye, triticale, barley, wheat, and maize grains (20 genotypes each), and 15 durum wheat and 13 pea grains. In addition, residues after ruminal incubation were included (at least from four samples per species for various incubation times). To establish CP and ST calibrations, 620 and 610 samples (grains and bag residues after incubation, respectively) were chemically analysed for their CP and ST concentration. Calibrations using wavelengths from 1250 to 2450 nm and the first derivative of the spectra produced the best results (R²_Validation=0.99 for CP and ST; standard error of prediction=0.47 and 2.10% DM for CP and ST, respectively). Hence, CP and ST concentration in cereal grains and peas and their bag residues could be predicted with high precision by NIRS for use in in situ studies. No differences were found between the effective ruminal degradation calculated from NIRS estimations and those calculated from chemical analyses (P>0.70). Calibrations were also calculated to predict ruminal degradation kinetics of cereal grains from the spectra of ground grains. Estimation of the effective ruminal degradation of CP and ST from the near-infrared spectra of cereal grains showed promising results (R²>0.90), but the database needs to be extended to obtain more stable calibrations for routine use.     

A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol,Nivalenol, and HT-2 Toxin in Cereal Matrices

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A K_m of 5.4 mM and a V_max of 16 μmol min⁻¹ mg⁻¹ were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.     

Standardization of an indirect PTA-ELISA for detection ofFusarium spp. in infected grains

Plant pathogenic fungi of the genusFusarium cause agriculturally important diseases of small grain cereals and maize. Especially the contamination of grains with the mycotoxin deoxynivalenol (DON) is harmful for animals and humans. For quantification of the severity ofFusarium head blight (FHB) in resistance evaluation and selection of cereals a fast, economical and reliable method is essential. Immunological methods appear to be particularly suitable for such an assessment. The results demonstrate that a selected antiserum was appropriate for the detection ofFusarium-exoantigens (ExoAg) in cereal grains by an indirect ELISA-format, although a discrimination between variousFusarium-species was not possible. The polyclonal antibody detection system was optimized for different parameters and a standard test protocol was elaborated. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004     

Determination of deoxynivalenol in cereals by HPLC-UV

A simple method for determination of deoxynivalenol (DON) in cereal samples is described. DON was extracted with methanol, the solvent evaporated, and the residue redissolved with water. This extract was purified on immunoaffinity columns. DON was determined by HPLC with UV-detection. The limits of detection (LOD) and quantification (LOQ) were 10 and 50 μg/kg, respectively. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

胶州湾夏秋季大气湿沉降中的营养盐及其入海的生态效应

为揭示大气湿沉降对胶州湾营养盐的输送通量及其生态效应,分别于2015年6—8月(夏季)、9—11月(秋季)采集胶州湾降水样品,测定了降水中不同形态N、P、Si的浓度。结果表明,降水中不同形态营养盐的浓度变化较大,且均与降水量呈负相关关系,其中NH4-N和NO3-N的浓度较高,溶解有机氮(DON)占溶解态总氮(DTN)含量的25.9%,而NO_2-N,PO_4-P和SiO_3-Si的浓度均很低。溶解无机氮(DIN)、DON、PO_4-P以及SiO_3-Si的湿沉降通量分别为141.7、61.87、0.35 mmol m~(-2)a~(-1)和0.12 mmol m~(-2)a~(-1)。受降水量和营养物质来源制约,各项营养盐湿沉降通量时间变化显著。农业活动导致的无机氮排放构成了胶州湾湿沉降DIN的主要来源。大气湿沉降DIN、DON、PO_4-P和SiO_3-Si分别占胶州湾总输入负荷的9.04%、10.24%、0.57%和0.17%,湿沉降输入的PO_4-P在夏、秋季分别可以支持0.575 mgC m~2d~(-1)和1.42 mg C m~2d~(-1)的新生产力;雨水中DIN/P比值高达1 617,突发性强降雨带来的营养盐输入会加剧表层水体的P限制和Si限制,对胶州湾浮游植物群落结构和粒级结构产生重要影响。大气湿沉降是胶州湾生源要素生物地球化学过程的重要一环,对营养物质收支的贡献及可能引发的生态效应不容忽视。     

An NMR-Based Metabolomic Approach to Investigate the Effects of Supplementation with Glutamic Acid in Piglets Challenged with Deoxynivalenol

Deoxynivalenol (DON) has various toxicological effects in humans and pigs that result from the ingestion of contaminated cereal products. This study was conducted to investigate the protective effects of dietary supplementation with glutamic acid on piglets challenged with DON. A total of 20 piglets weaned at 28 d of age were randomly assigned to receive 1 of 4 treatments (5 piglets/treatment): 1) basal diet, negative control (NC); 2) basal diet +4 mg/kg DON (DON); 3) basal diet +2% (g/g) glutamic acid (GLU); 4) basal diet +4 mg/kg DON +2% glutamic acid (DG). A 7-d adaptation period was followed by 30 days of treatment. A metabolite analysis using nuclear magnetic resonance spectroscopy (¹H-NMR)-based metabolomic technology and the determination of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities for plasma, as well as the activity of Caspase-3 and the proliferation of epithelial cells were conducted. The results showed that contents of low-density lipoprotein, alanine, arginine, acetate, glycoprotein, trimethylamine-N-oxide (TMAO), glycine, lactate, and urea, as well as the glutamate/creatinine ratio were higher but high-density lipoprotein, proline, citrate, choline, unsaturated lipids and fumarate were lower in piglets of DON treatment than that of NC treatment (P<0.05). Compared with DON treatment, dietary supplementation with glutamic acid increased the plasma concentrations of proline, citrate, creatinine, unsaturated lipids, and fumarate, and decreased the concentrations of alanine, glycoprotein, TMAO, glycine, and lactate, as well as the glutamate/creatinine ratio (P<0.05). Addition glutamic acid to DON treatment increased the plasma activities of SOD and GSH-Px and the proliferating cell nuclear antigen (PCNA) labeling indexes for the jejunum and ileum (P<0.05). These novel findings indicate that glutamic acid has the potential to repair the injuries associated with oxidative stress as well as the disturbances of energy and amino acid metabolism induced by DON.     

Growth stimulation of Microcystis aeruginosa by a bacterium from hyper-eutrophic water (Taihu Lake, China)

Cyanobacteria are the causative organisms of the algal blooms that occur in Taihu Lake. Dissolved organic nitrogen (DON) comprises a significant composition of nitrogen (N) pool in the water and may increase the nutrient source of microalgae. In the present study, we investigated the relationship between Microcystis aeruginosa, Pseudomonas sp. A3CT isolated from Taihu, and DON compounds. Co-incubation (3 days) of the bacterium with six DON compounds (four free amino acids and two combined amino acids) was collected as six decomposed DON solutions. The decomposed DON solutions of six compounds were used to test the stimulatory effect of nutrient regeneration by the bacterium. The growth of M. aeruginosa was significantly enhanced by the six decomposed DON solutions. M. aeruginosa grew much better under the six decomposed DON solutions than the corresponding undigested DON forms. Especially, the decomposed l-lysine solution, not only avoided the inhibiting effect of lysine on M. aeruginosa, but significantly promoted the cyanobacterial growth. Further chemical tests indicated that A3CT transformed DON into NH₄ ⁺, which was utilized by M. aeruginosa. These results demonstrate that the bacterium plays an important role in decomposing unavailable DON forms into available NH₄ ⁺, which suggests that the bacterium contributes to the fast growth of M. aeruginosa. Moreover, this phenomenon, in conjunction with previous studies, indicates that the responsible and effective way of harmful blooms is reducing the N and P inputs (including DON and DOP).     

Mycotoxins in several Polish food products in 2004–2005

In 2004–2005, samples of several selected Polish foods such as cereal products, nuts, dried fruits, coffee and culinary spices collected from Warsaw market and taken from food producers were analyzed on presence of aflatoxin B₁, B₂, G₁, G₂ (AF), ochratoxin A (OTA), zearalenone (ZEA) and deoxynivalenol (DON). After extraction and clean-up of extracts on immunoaffinity columns (IAC), mycotoxin analyses were carried out by HPLC using fluorescence and UV detectors. The concentrations of aflatoxins and ochratoxin A depending on the kind of sample ranged from 0.02 to 7.8 (one sample, of peanuts) and 0.02–11.9 μg/kg (one coffee sample), respectively. The levels of ZEA and DON were found to be below 50 °g/kg.     

Modified use of a commercial ELISA kit for deoxynivalenol determination in rice and corn silage

The analysis of deoxynivalenol (DON) in silage samples using enzyme-linked immunosorbent assay (ELISA) often leads to an overestimation. To better analyze DON in rice and corn silages using a commercially available ELISA kit, a cleanup method using a MultiSep #226 column was developed. As a result, overestimation of DON by the influence of specific cross-reaction with acetyldeoxynivalenol (AcDON) was confirmed. In samples where AcDON was not detected by liquid chromatography with mass spectrometry (LC-MS), no samples showed a significant difference (P?<?0.05) in DON amounts between ELISA with cleanup and LC-MS analysis. For the recovery study, blank silage was spiked with 0.5 or 1.0 mg/kg DON. The mean recoveries of DON determined by ELISA with cleanup and LC-MS analysis were 112 and 96 %, respectively, and the relative standard deviation for the repeatability (RSD_r) were 8.2 and 9.8 %, respectively. No samples showed a significant difference (P?<?0.05) in DON concentration determined by either ELISA or LC-MS analysis. A collaborative study to validate this rapid method was carried out using four samples, two rice and two corn silage, by 10 participating laboratories. Each sample was analyzed using blind duplicates. The mean values of DON detected were 1.5–2.3 mg/kg, RSD_r and the relative standard deviation for the reproducibility (RSD_R) were 4.1–12.7 and 7.6–23.4 %, respectively, and the HorRat values were 0.5–1.6. Therefore, the overestimation of DON by the influence of nonspecific cross-reaction with sample matrix was reduced by the cleanup method using a MultiSep #226 column, and analysis of DON in silage was improved. This use of this method for estimation of DON contamination in silage allows rapid detection at the place of use that is likely to result in improved animal health.