Verhalten von Mykotoxinen bei der Ethanolerzeugung aus Getreide

By means of a modelling experiment we followed the fate of deoxynivalenol (DON) and zearalenone (ZEA) in the production of bioethanol from grain. The experiment has been performed at the ‘Versuchs- und Lehranstalt für Brauerei in Berlin e.V.’ A total of 33 kg. triticale with a concentration of 3.2 mg/kg DON and 0.035 mg/kg ZEA were treated with water and enzymes which yielded the mash. By adding yeasts the mixture was then brought to formentation. Finally, the alcohol was obtained by destillation. The residue was dried by centrifugation to a content of 25% dry matter and put in silage tubes. In each part of the whole process test samples were taken and the content of DON and ZEA was determined. After three month the silage was scraped out from the tubes and analyzed, too. The mycotoxins were analyzed by HPLC with DAD and HPLC with fluorescence detection, corresponding to VDLUFA-methods. The balance of the mycotoxin contents showed no loss of DON in the process. For ZEA, however, a deficit of about 35% was noted. Because of the mass loss during starch fermentation (33 kg grain yield 8 kg draff) the mycotoxin contents were enhanced by a factor of 2 to 4. DON is well dissolved in water and therefore partially washed out by filtration of the mash. As the whole process is performed in practice as a cycle, the filtrate with DON is permanently recycled in the brewery process. Therefore the mycotoxin concentration in the original grain should not exceed the legal EU boundary values of 1.25 mg/kg DON and 0.1 mg/kg ZEA.     

Mycotoxins inactivation by extrusion cooking of corn flour

AIMS: To evaluate the effects of the extrusion cooking process on the inactivation of mycotoxins in corn flour. METHODS AND RESULTS: Samples of corn flour experimentally contaminated with aflatoxin B1 (AFB1) (50 ppb) and deoxynivalenol (DON) (5 ppm) were extruded. The effects of three extrusion variables (flour moisture, extrusion temperature and sodium metabisulphite addition) were analysed according to a two-level factorial design. The process was effective for the reduction of DON content (higher than 95%) under all the conditions assessed, but was only partially successful (10-25%) for the decontamination of AFB1. CONCLUSION: Extrusion cooking is effective for the inactivation of DON but is of limited value for AFB1, even if metabisulphite is added. More severe extrusion conditions are needed for the detoxification of AFB1. SIGNIFICANCE AND IMPACT OF THE STUDY: As contamination with DON occurs mainly in the field prior to harvesting and that of AFB1 is normally produced during grain storage, maize is often contaminated with DON but not with AFB1. Under these conditions, the described extrusion process can be used for the detoxification of DON. The addition of sodium metabisulphite did not significantly affect the inactivation of AFB1. Extrusion cooking is therefore an appropriate treatment for vomitoxin-contaminated maize in countries where, because of the prevailing conditions, these are the only toxins present.     

Natural Occurrence of 16 Fusarium Toxins in Grains and Feedstuffs of Plant Origin from Germany

A total of 220 samples comprising cereals, cereal byproducts, corn plants and corn silage as well as non-grain based feedstuffs was randomly collected during 2000 and 2001 from sources located in Germany and analysed for 16 Fusarium toxins. The trichothecenes scirpentriol (SCIRP), 15-monoacetoxyscirpenol (MAS), diacetoxyscirpenol (DAS), T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), neosolaniol (NEO), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivealenol (15-ADON), nivalenol (NIV) and fusarenon-X (FUS-X) were determined by gas chromatography/mass spectrometry. Zearalenone (ZEA) and α- and β-zearalenol (α- and β-ZOL) were analysed by high performance liquid chromatography with fluorescence and UV-detection. Detection limits ranged between 1 and 19 μg/kg. Out of 125 samples of a group consisting of wheat, oats, corn, corn byproducts, corn plants and corn silage only two wheat samples did not contain any of the toxins analysed. Based on 125 samples the incidences were at 2–11% for DAS, NEO, T-2 Triol, FUS-X, α- and β-ZOL, at 20–22% for SCIRP, MAS, T-2 tetraol and 3-ADON, at 44–74% for HT-2, T-2, 15-ADON, NIV and ZEA, and at 94% for DON. Mean levels of positive samples were between 6 and 758 μg/kg. Out of 95 samples of a group consisting of hay, lupines, peas, soya meal, rapeseed meal and other oilseed meals, 64 samples were toxin negative. DAS, T-2 triol, NEO and FUS-X were not detected in any sample. The incidences of DON and ZEA were at 14 and 23% respectively, those of the other toxins between 1–4%, mean levels of positive samples were between 5 and 95 μg/kg.     

Reduction of Soybean Meal Non-Starch Polysaccharides and ��-Galactosides by Solid-State Fermentation Using Cellulolytic Bacteria Obtained from Different Environments

Soybean meal (SBM) is an important protein source in animal feed. However, the levels of SBM inclusion are restricted in some animal species by the presence of antinutritional factors (ANFs), including non-starch polysaccharides (NSPs) and α-galactosides (GOSs). The aim of this study was to reduce the soybean meal NSPs and GOSs by solid-state fermentation (SSF) using a combination of cellulolytic bacteria isolated from different environments (termites, earthworms, corn silage and bovine ruminal content). To analyse the key enzymatic activities, the isolates were grown in minimal media containing NSPs extracted from SBM. The selected bacterial strains belonged to the genera Streptomyces, Cohnella and Cellulosimicrobium. SSF resulted in a reduction of nearly 24% in the total NSPs, 83% of stachyose and 69% of raffinose and an increase in the protein content. These results suggest that cellulolytic bacteria-based SSF processing facilitates SBM nutritional improvement. In addition, the use of fermented SBM in animal diets can be recommended.     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination.

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

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.     

Metabolisierung von Deoxynivalenol beim Schwein: Bestimmung von DON und DOM-1 im Urin vom Schwein

This paper describes a feeding study with 7 pigs, which were fed with deoxynivalenol contaminated oats at a level of 0.23 mg/kg body mass/day over 16 experiment days. The contamination level of consumed feed was 14.4 mg DON/kg in the ration. The parallel control group of 7 pigs were fed with DON free oats. Urine samples were taken each two days. The content of DON and DOM-1 (de-epoxy deoxynivalenol) in urine was determined. The mean concentration of DON in urine of animals in trial group was 580 μg/l, whereas DOM-1 32 μg/l.     

Wirkung von Deoxynivalenol beim wachsenden Schwein in Abhängigkeit von der Darreichungsform

To evaluate the minimum effective dose of pure DON leading to measurable losses in weight gain and feed consumption a special feeding experiment was created to compare the effect of DON in natural contaminated wheat and — for the first time — a non-cereal diet (potato) spiked with pure DON. Examined parameters were weight gain, feed consumption and blood parameters.Three trials were conducted. In the first trial a concentration of 4000 µg DON/kg feed was used. In the second and third trial we used concentrations of 4000 and 6000 µg DON/kg feed. Severe effects on feed consumption and weight gain were found only in the second trial (naturally contaminated wheat ad lib.). By contrast, no differences in any parameter were found in the first (restricted feeding) and third trial (non-cereal diet spiked with pure DON ad lib.).     

玉米浆对玉米粉和木薯淀粉发酵甘油的影响及二者的比较

以玉米粉和木薯淀粉为原料 ,比较了二者的液化和糖化 ,结果表明 :在相同条件下 ,木薯淀粉液化时间较短 ,玉米粉液化时间较长 ,但二者的液化液均较易糖化。然后分别以玉米粉和木薯淀粉糖化液为原料 ,用耐高渗酵母发酵生产甘油 ,研究了玉米浆对二者甘油发酵的影响并对二者进行了比较 ,结果表明 :当玉米粉和木薯淀粉糖化液还原糖含量分别为 2 5 % ,尿素为 0 .2 % ,pH为 4 .5时 ,用玉米粉糖化液发酵甘油时可不添加玉米浆 ,甘油产量最高可达 2 % ,而用木薯淀粉糖化液发酵甘油时 ,适宜的玉米浆为 0 .15 % ,甘油产量最高可达 4 .9%。对二者的比较结果表明 :用玉米粉糖化液为发酵原料时 ,发酵时间较短 ,残糖降低较快 ,甘油产量较低 ,在 36h之后 ,甘油开始反耗。而用木薯淀粉糖化液发酵时 ,发酵时间较长 ,残糖降低较慢 ,甘油产量较高 ,在 72h之后 ,甘油开始反耗。     

Effects of pressed beet pulp silage inclusion in maize-based rations on performance of high-yielding dairy cows and parameters of rumen fermentation

Beet pulp contains high amounts of pectins that can reduce the risk of rumen disorders compared to using feedstuffs high in starch. The objective was to study the effects of inclusion of ensiled pressed beet pulp in total mixed rations (TMR) for high-yielding dairy cows. Two TMR containing no or about 20% (on dry matter (DM) basis) beet pulp silage were used. The beet pulp silage mainly replaced maize silage and corn cob silage. The TMR were intentionally equal in the concentrations of energy and utilisable crude protein (CP) at the duodenum. TMR were fed to 39 and 40 dairy cows, respectively, for 118 days. The average daily milk yield was about 43 kg/day. No significant differences in milk yield and milk fat or milk protein content were detected. DM intake of cows was significantly reduced by the inclusion of beet pulp silage (23.0 v. 24.5 kg/day). However, a digestibility study, separately conducted with sheep, showed a significantly higher organic matter digestibility and metabolisable energy concentration for the TMR that contained beet pulp silage. In vitro gas production kinetics indicated that the intensity of fermentation was lower in the TMR that contained beet pulp silage. In vitro production of short-chain fatty acids, studied using a Rusitec, did not differ between the TMR. However, the inclusion of beet pulp silage in the ration caused a significant reduction in the efficiency of microbial CP synthesis in vitro. The amino acid profile of microbial protein remained unchanged. It was concluded that beet pulp silage has specific effects on ruminal fermentation that may depress feed intake of cows but improve digestibility. An inclusion of beet pulp silage of up to 20% of DM in rations for high-yielding dairy cows is possible without significant effects on milk yield and milk protein or milk fat.     

The bacterial community and metabolome dynamics and their interactions modulate fermentation process of whole crop corn silage prepared with or without inoculants

Multi-omics approach was adopted to investigate the modulation of bacterial microbiota and metabolome as well as their interactions in whole crop corn ensiling systems by inoculating homofermentative Lactobacillus plantarum or heterofermentative Lactobacillus buchneri. Inoculations of the two different inoculants resulted in substantial differences in microbial community and metabolic composition as well as their dynamics in ensiled corn. Inoculants also altered the correlations of microbiota in different manners, and various keystone species were identified in corn silages with different treatments. Many metabolites with biofunctional activities like bacteriostatic, antioxidant, central nervous system inhibitory and anti-inflammatory were found in the present silage. A constitutive difference in microbiota dynamics was found for several pathways, which were upregulated by specific taxa in middle stage of fermentation, and widespread associations between metabolites with biofunctions and the species of lactic acid bacteria dominated in silage were observed. Multiple microbial and metabolic structures and dynamics were correlated and affected the fermentation process of the corn ensiling systems. Results of the current study improve our understanding of the complicated biological process underlying silage fermentation and provide a framework to re-evaluate silages with biofunctions, which may contribute to target-based regulation methods to produce functional silage for animal production.     

Comparative denaturing gradient gel electrophoresis analysis of fungal communities associated with whole plant corn silage.

Significant portions of grain produced for livestock consumption are convened into ensiled forage. Silage producers have long recognized the positive effects of using an inoculant to insure the proper transformation of forage into a palatable and digestible feedstuff. When silage is fed from a storage structure, exposure to air stimulates the growth of epiphytic aerobes that may result in the loss of up to 50% of the dry matter. Moreover, fungi have been found to be associated with ensiled forage, but their growth is normally suppressed by the anaerobic conditions. However, the introduction of oxygen results in a fungal bloom, and the fungi and the associated metabolites may result in lost productivity in the livestock consuming the contaminated forage. In this study, we report on the diversity of the fungal community associated with whole plant corn silage during the ensiling process, and the effect of two different bacterial inoculants as compared with the uninoculated natural epiphytic fermentation on the distribution of the fungi associated with the silage. The fungal community from duplicate mini-silo packages of the same treatment was analyzed by denaturing gradient gel electrophoresis and direct sequencing of the resulting operational taxonomic units. This method proved useful in analyzing the complex microbial communities associated with the forage in that it was possible to determine that one inoculant dramatically influenced the fungal community associated with whole plant corn silage.     

Silage and animal health

The process of preserving crops by fermentation in silos is under the control of the farmer to a much lesser degree compared to the level of control by the manufacturer over the production of other fermented foods, such as cheese and yoghurt. Additives designed to direct the extent and pattern of the fermentation are relatively unpopular in most countries, and their use is not guaranteed to remove the risk of undesirable components in silage. Hazards to animal health associated with silage fall into three categories: (1) undesirable micro-organisms e.g. Listeria, enterobacteria, clostridia and moulds; (2) undesirable chemicals, e.g. mycotoxins, and (3) excess acidity and other metabolic disorders. In some regions of Europe, the production of silage is discouraged or prohibited because of the risk of undesirable microbes. The princIpal risk in these areas is that of the secondary fermentation of cheese made from milk contaminated by bacterial spores, rather than a direct hazard of contaminated silage to animal health. With the possible exception of high dry matter silage conserved in large bales, respiratory hazards to animals from moulds and their spores generally are less from silage than hay. Mycotoxins and phytoestrogens may survive the ensiling period and constitute risks to animal health. Relatively little is known about the epidemiology of diseases that may be linked to undesirable chemicals and excess acidity in silage. Therefore, research is needed to define epidemiologically and mechanistically the risks to animal health and to the human food chain from silages contaminated with pathogenic bacteria and mycotoxins, and to understand more completely the relationships between the physical and chemical compositions of silage and metabolic disorders in animals.     

Microbial inoculant effects on silage and in vitro ruminal fermentation, and microbial biomass estimation for alfalfa, bmr corn, and corn silages

Whole crop third cut alfalfa, brown mid-rib (bmr) corn, and corn were chopped and inoculated with one of four microbial inoculants used. Uninoculated silage was the control treatment. Each crop was ensiled in four mini-silos (1 L glass jars) per treatment. All silos were fermented for 60 days at room temperature (22 °C), and then they were opened and analyzed for fermentation products, fiber constituents and N fractions. A fraction of wet silage was ground with a blender for 30 s. In vitro gas production was measured in 160 ml sealed serum vials at 3, 6, 9, 24, and 48 h using the wet ground silage. At 9 and 48 h, rumen fluid was analyzed for volatile fatty acids (VFA) and microbial biomass yield (MBY). In all the three crops, the four inoculants produced only minor changes in pH and fermentation products during ensiling. Of the variables measured, soluble nonprotein N fractions were the characteristics most often affected by some inoculants. At 9 h incubation, in vitro gas production and VFA did not differ between control and inoculated silages, but MBY did. Among crops, alfalfa and corn silages had higher MBY than did bmr corn silage. Among inoculants, three of the four inoculated silages produced more MBY than did control. At 48 h, alfalfa silage produced higher MBY than did corn or bmr silage, and two of the inoculated silages had more MBY than did the control. There was no inoculant by crop interaction. Results suggest that some silage inoculants are capable of altering rumen fermentation, even in cases where effects on silage fermentation are small, and that this effect may be linked to better preservation of crop protein during ensiling.     

A survey of Fusarium toxins in Austrian feed stuffs

During the last decade observations have been made concerning the occurrence of the fusariotoxins zearalenone and deoxynivalenol in Austria. Feed samples were tested in a large-scaled examination. They usually consisted of cereals (barley, oat, wheat, rye and corn), mixed feed (meal or pellets) and corn or corn cob silage.     

A review on comparative data concerning <Emphasis Type="Italic">Fusarium</Emphasis> mycotoxins in Bt maize and non-Bt isogenic maize

The European corn borer reportedly promotes the infection of maize by Fusarium spp. Stalk and ear rots caused by Fusarium spp. are often related to mycotoxin accumulation in maize kernels. As a result, food and animal feed from maize are more severely contaminated with Fusarium mycotoxins: e.g. fumonisins (FUM), deoxynivalenol (DON) and zearalenone (ZEA). Bt maize is primarily an important potential tool for insect pest protection, both in the European Union and in other countries. Bt maize carrying the Bt genes is highly resistant to European corn borer larval feeding due to Bt toxin (δ toxin) production. Effective measures to combat pests therefore often have a positive side-effect in that they also reduce mycotoxin levels. Comparative analysis was used to the evaluation of the studies dealing with the reduction of Fusarium mycotoxins in Bt maize. Nineteen out of 23 studies on Bt maize came to the conclusion that Bt maize is less contaminated with mycotoxins (FUM, DON, ZEA) than the conventional control variety in each case.     

农作物秸秆体外发酵营养特性及其组合利用研究

采用体外产气技术,研究了不同粗饲料及其组合的体外发酵特性,探讨了合理利用不同粗饲料的有效途径.结果表明,玉米秸秆青贮后其理论最大产气量及产气速率比玉米秸分别高19.1%和8.2%.组合秸秆的理论产气量及产气速率要高于单一秸秆,发酵48 h后,组合秸秆的产气量可获得显著的正组合效应.可以认为,青贮玉米秸秆及不同粗饲料间进行组合可有效提高其营养特性.粗料间进行组合时,以青贮玉米秸与小麦秸或稻秆按25∶75和50∶50的比例组合较为适宜.     

Influence of the soya meal fractions on gibberellic acid and gibberellin A production in submerse cultivation ofGibberella fujikuroi

The relationship of different soya meal components to gibberellin (GA) production was studied. Fluorometric assay confirmed that under the given fermentation conditions, only gibberellic acid (GA₃) was synthesized on medium containing corn steep. On substituting soya flour for corn steep, the same amount of GA₃ was produced and in addition gibberellin A (GA₁) was formed. The GA₃: GA₁ ratio was 1∶1. The course of fermentation in media containing the soya meal protein fraction (fraction I), the soya meal amino acid complex, the corn steep amino acid complex and individual amino acids (γ-aminobutyric acid or tryptophane) was the same as in the control medium containing soya meal. The soya meal fraction II, which is characterized by a high cellulose and carbohydrate content, raised GA production by 25% as compared with production in medium containling soya meal; it simultaneously stimulated GA₃ production, so that the final GA₃: GA₁ ratio was 4∶1.