Production of zearalenone,moniliformin and trichothecenes in intact sugar beets under laboratory conditions

Five toxigenic isolates of Fusarium species were tested for the production of zearalenone, moniliformin and trichothecenes (deoxynivalenol, 15-acetyldeoxynivalenol, T-2, HT-2 and neosolaniol) when grown on solid sugar beet slices in the laboratory for thirty days. The isolates were also grown on a solid rice medium for comparison. High zearalenone and trichothecene-producing isolates originally obtained from corn and corn-based feedstuff were compared with isolates obtained from sugar beets. One moniliformin-producing isolate from wheat was included in the study. With the exception of moniliformin, all toxins were produced on both substrates; however, the rice medium yielded the greater concentrations except for HT-2 which was produced on sugar beets in equal or greater concentrations. Zearalenone production on rice reached 729–1943 gmg/g whereas on sugar beet it reached 72–193 gmg/g. The moniliformin-producing isolate grew well on both substrates; however, moniliformin was produced only on the rice substrate. This study demonstrates for the first time that Fusarium species can produce both zearalenone and the trichothecenes on a sugar beet substrate.     

Studies on extraction of fumonisins from rice, corn-based foods and beans

Different solvent mixtures were examined for extraction of fumonisins from various naturally contaminated and spiked foods and foodstuffs: rough rice, retail rice, rice flour, white corn flour, corn meal, corn starch, corn flakes, tortilla/corn chips, white bean flour, white beans, mung beans, adzuki beans and infant cereals. Most of the naturally contaminated samples were analyzed using the extraction solvent mixtures methanol-acetonitrile-water (25:25:50) (solvent A) and methanol-water (75:25 or 80:20) (solvents B, BB); some were extracted with 0.1 M sodium hydrogen phosphate-acetonitrile (1:1, adjusted to pH 3.0 with o-phosphoric acid) (solvent C) and methanol-0.025 M borate buffer (3:1, adjusted to pH 9.2 with 1 N sodium hydroxide) (solvent D). A 1-ml SAX solid phase extraction column was used for the cleanup in all cases except for infant cereals, for which immunoaffinity chromatography was used; fumonisin concentrations were determined by liquid chromatography. Solvent A gave slightly better extraction of fumonisins from one of two samples of naturally contaminated rough rice than solvent B (fumonisin B₁: 4080 ng/g versus 3150 ng/g; fumonisin B₂:1100 ng/ g versus 922 ng/g) and much better extraction than solvent C (1210 ng/g fumonisin B₁ and 315 ng/g fumonisin B₂) or solvent D (372 ng/ g fumonisin B1 and 191 ng/g fumonisin B2). However, spike recoveries on a similar rice naturally contaminated at a lower level were only in the 43–53% range (solvent A). Recovery of fumonisins was very poor from spiked white rice flour but satisfactory from other rice foods. Solvent A similarly gave slightly better extraction of fumonisins from a sample of naturally contaminated white corn flour than solvent B (fumonisin B₁ 1260 ng/g versus 931 ng/g; fumonisin B₂: 511 ng/g versus 447 ng/g ) and better extraction than solvents C and D. Solvent A was also a better solvent for extraction of fumonisins from naturally contaminated tortilla chips and infant cereals. Study of naturally contaminated corn starch was confounded by instability of fumonisins in this food. Recovery of fumonisins from spiked corn meal, tortilla chips, corn flakes, various types of beans and infant cereals with solvent A and/or solvent B (or BB) was satisfactory.     

Comparison of commercially available kits with standard methods for detection of Salmonella strains in foods.

Six commercial kits were compared with the U.S. Food and Drug Administration (USFDA) method and the Japanese standard method for Salmonella isolation in foods. When only Salmonella serovars were tested, many of the methods performed well; however, when foods were artificially inoculated, only the USFDA method and immunomagnetic separation coupled with the xylose-lysine-brilliant green agar method (MS-XLBG) could positively detect Salmonella serovars. All seven wild-type Salmonella serovars were detected by the USFDA method, and the MS-XLBG method detected salmonellae from six samples.     

Occurrence of trichothecenes, zearalenone and ochratoxin A in cereals and mixed feed from central Lithuania

A total of 92 samples — 23 winter wheat, 12 summer barley, 5 oats and 52 mixed feed — were collected from a state factory in Kaunas, Lithuania and were analysed for the presence of trichothecenes, zearalenone (ZEN) and ochratoxin A (OA) using gas chromatography with electron capture detection and immunoaffinity column/high performance liquid chromatography with fluorescence and UV detections. Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin and HT-2 toxin were detected at concentrations above 10 μg/kg in 68%, 48%, 38% and 8% of cereal samples, respectively, and in 98%, 88%, 12% and 8% of samples of mixed feed for swine and poultry. More than 10 μg/kg of zearalenone and ochratoxin A were found in 58% and 92% of the mixed feed samples, respectively. The highest concentrations of all analysed trichothecenes in Lithuanian mixed feed and cereal grains, with an exception of T-2 toxin in one oat lot and one sample of mixed feed and OA in two mixed feed samples, were lower than those reported as Lithuanian advisory or tolerance limits.     

Chemotaxonomy of Gibberella zeae with special reference to production of trichothecenes and zearalenone

By adopting a single-spore isolation technique, 113 isolates of Gibberella zeae, the perfect stage of Fusarium graminearum, were isolated from rice stubbles in barley and wheat fields and tested for production of trichothecenes and zearalenone on rice grains. Of the isolates, 93% produced the trichothecenes, and they could be subdivided into two chemotaxonomic groups: nivalenol and fusarenon-X producers and deoxynivalenol and 3-acetyldeoxynivalenol producers. No cross production of these two types of trichothecenes was observed in these isolates. Zearalenone was detected in 68% of the isolates, but no clear relationship could be observed regarding its position with respect to the two chemotaxonomic groups.     

Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods.

Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.     

Chemotaxonomy of Gibberella zeae with special reference to production of trichothecenes and zearalenone.

By adopting a single-spore isolation technique, 113 isolates of Gibberella zeae, the perfect stage of Fusarium graminearum, were isolated from rice stubbles in barley and wheat fields and tested for production of trichothecenes and zearalenone on rice grains. Of the isolates, 93% produced the trichothecenes, and they could be subdivided into two chemotaxonomic groups: nivalenol and fusarenon-X producers and deoxynivalenol and 3-acetyldeoxynivalenol producers. No cross production of these two types of trichothecenes was observed in these isolates. Zearalenone was detected in 68% of the isolates, but no clear relationship could be observed regarding its position with respect to the two chemotaxonomic groups.     

Natural occurrence of Fusarium mycotoxins (trichothecenes and zearalenone) in barley and corn in Korea.

Barley is produced in four provinces, Chonbuk, Chonnam, Kyungbuk, and Kyungnam, and corn is mainly produced in the Kangwon province in Korea. The natural occurrence of Fusarium mycotoxins was surveyed in 39 barley and 46 corn samples from different areas. Five 8-ketotrichothecenes, namely deoxynivalenol (DON), nivalenol (NIV), 4-acetylnivalenol (4-ANIV), 3-acetyldeoxynivalenol (3-ADON), and 4,15-diacetylnivalenol (4,15-DANIV), and zearalenone (ZEA) were detected in barley. DON, NIV, and ZEA were the major contaminants in barley, with mean levels of 170, 1,011, and 287 ng/g, respectively. On the other hand, DON, 15-acetyldeoxynivalenol (15-ADON), NIV, 4-ANIV, 4,15-DANIV, and ZEA were detected in corn samples. DON and 15-ADON were the major contaminants in corn, with mean levels of 310 and 297 ng/g, respectively. The survey indicated that the natural occurrence of monoacetyl-DON and the ratios of NIV to DON in two cereals were different. In addition, this is the first report of the natural occurrence of 4,15-DANIV in cereals.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Mechanism and use of the commercially available viability stain, BacLight.

BACKGROUND: BacLight (Molecular Probes, Eugene, OR, USA) is a popular fluorescence-based two-component stain for determining bacterial cell viability. The main purpose of this work was to fully elucidate the mechanism and to determine why it is sometimes reported that cells stain simultaneously live and dead. METHODS: Solutions of DNA were stained with the two components, propidium iodide (PI) and SYTO9, in different combinations, and fluorescence spectra were collected. RESULTS: K(PI) and K(SYTO9) were approximately 3.7 x 10(5)/M and 1.8 x 10(5)/M. SYTO9 emissions were stronger and overlapped those of PI. Fluorescence resonance energy transfer from SYTO9 to PI was observed. It was, even under normal conditions, possible for DNA bound SYTO9 to have a component in the red region equal to that of DNA bound PI. Potentially confusing emissions were also found to occur when PI was not in sufficient excess to saturate nucleic acid (>0.4 M PI to 1 M DNA base pairs). CONCLUSIONS: The mechanism is a combination of displacement of SYTO9 by PI and quenching of SYTO9 emissions by fluorescence resonance energy transfer. Confusing results can occur if the relative intensities of the stains or the concentration of PI relative to nucleic acid are not properly accounted for.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Compressive and shear properties of commercially available polyurethane foams

BACKGROUND: The shear properties of rigid polyurethane (PU-R) foams, routinely used to simulate cancellous bone, are not well characterized. METHOD OF APPROACH: The present assessment of the shear and compressive properties of four grades of Sawbones "Rigid cellular" PU-R foam tested 20 mm gauge diameter dumb-bell specimens in torsion and under axial loading. RESULTS: Shear moduli ranged from 13.3 to 99.7 MPa, shear strengths from 0.7 MPa to 4.2 MPa. Compressive yield strains varied little with density while shear yield strains had peak values with "200 kgm-3" grade. CONCLUSIONS: PU-R foams may be used to simulate the elastic but not failure properties of cancellous bone.     

Evaluation of commercially available heart rate monitors.

The accuracy and tracking ability of nine commercially available heart rate monitors were assessed. The heart rate of 16 young healthy men was continuously monitored by a single-lead electrocardiograph while they exercised on a stationary bicycle ergometer. Readings were obtained from the devices during exercise. The devices that measured the cardiac electrical potential with a three-electrode system or that incorporated a light transmission device attached to the earlobe were the most accurate and provided suitable monitoring of the heart rate during exercise.     

Structural and nonstructural carbohydrate,fat, and protein composition of commercially available,whole produce

Previously reported values for produce items often reflect only the human edible portion although animals generally eat the entire item. Produce can comprise a significant proportion of a captive, exotic animal's diet; therefore, nutrient values based on whole items will enable a more accurate diet formulation. Whole produce items, including fruits, vegetables, and leafy green vegetables, were analyzed for dry matter, neutral detergent fiber, acid detergent fiber, crude protein, fat, ash, pectin, fructan, and free sugar concentrations. The free sugars were typed and quantified. As expected, the produce contained low concentrations of neutral detergent fiber, averaging 13.4% for fruits, 18.8% for vegetables, and 21.5% for leafy green vegetables/other items on a 100% dry matter basis. Produce ranged substantially in structural and nonstructural carbohydrates, protein, fat, and free‐sugar concentration. Free‐sugar ratios of glucose, fructose, and sucrose varied among items. This information can be used for more accurate formulation of zoological diets. Zoo Biol 0:1–15, 2005. © 2005 Wiley‐Liss, Inc.     

Mycotoxins (trichothecenes, zearalenone and fumonisins) in cereals associated with human red-mold intoxications stored since 1989 and 1991 in China

Two corn powder samples implicated in the human food poisoning that occurred in Guangxi province in 1989, and eight wheat and two barley samples linked to an episode that involved about 130,000 people in gastrointestinal disorders in Anhui province in 1991 were analyzed for trichothecenes including deoxynivalenol (DON), nivalenol (NIV) and their esters, zearalenone (ZEA) and fumonisins (FMs) by gas chromatography/mass spectroscopy and high performance liquid chromatography, and T-2 toxin by enzyme-linked immunosorbent assays. DON was detected in all samples as a major trichothecene (16-51,450 microg kg(-1)), and NIV was in one corn, one barley and all wheat at relatively low levels (10-6935 microg kg(-1)). ZEA was found in all corn and barley, and six wheat samples (46-3079 microg kg(-1)). In addition, 3-acetyl-DON (2544 microg kg(-1)) and 15-acetyl-DON (2537 microg kg(-1)) were detected separately in one corn and one wheat sample. The highest levels of these mycotoxins were found in one wheat sample associated with the human intoxication in Anhui province. FMs in corn were below 1000 microg kg(-1). Risks of DON and ZEA on the people who consumed the causative cereals were assessed.