Volatile compounds of Aspergillus strains with different abilities to produce ochratoxin A

Volatile compounds emitted by Aspergillus strains having different abilities to produce ochratoxin A were investigated. Thirteen strains of Aspergillus ochraceus, three belonging to the A. ochraceus group, and eight other species of Aspergillus were examined for their abilities to produce volatile compounds and ochratoxin A on a wheat grain medium. The profiles of volatile compounds, analyzed using SPME, in all A. ochraceus strains, regardless of their toxeginicity, were similar and comprised mainly of 1-octen-3-ol, 3-octanone, 3-octanol, 3-methyl-1-butanol, 1-octene, and limonene. The prevailing compound was always 1-octen-3-ol. Mellein, which forms part of the ochratoxin A molecule, was found in both toxigenic and nontoxigenic strains. Volatile compounds produced by other Aspergillus strains were similar to those of A. ochraceus. Incubation temperatures (20, 24, and 27 degrees C) and water content in the medium (20, 30, and 40%) influenced both volatile compounds formation and ochratoxin A biosynthesis efficiency, although conditions providing the maximum amount of volatiles were different from those providing the maximum amount of ochratoxin A. The pattern of volatiles produced by toxigenic A. ochraceus strains does not facilitate their differentiation from nontoxigenic strains.     

Effect of ochratoxin A-producing Aspergilli on stilbenic phytoalexin synthesis in grapes

Berries of Vitis vinifera L. cv. Barbera were infected, at veraison and during ripening, by a conidial suspension of A. japonicus, A. ochraceus, A. fumigatus and two isolates of A. carbonarius to control ochratoxin A production and stilbene induced synthesis. The experimental design provided also for intact and punctured berries and incubation temperature of 25 degrees C and 30 degrees C. All the tested fungi, except A. fumigatus, significantly increased trans-resveratrol synthesis over the control, while trans-piceid was not affected; only A. ochraceus significantly elicited the berries to synthesize piceatannol. The two isolates of A. carbonarius produced higher amounts of ochratoxin A than did the other fungi. A positive correlation between ochratoxin A and trans-resveratrol synthesis occurred. trans-Resveratrol and piceatannol showed fungicidal activity against A. carbonarius, being able to completely inhibit fungal growth at a concentration of 300 microg/g and 20 microg/g, respectively.     

Biodegradation of ochratoxin A by fungi isolated from grapes

Ochratoxin A is a mycotoxin present in several food products for which levels should be reduced. Chemical, physical, and biological methods have been proposed for the detoxification of mycotoxins, biological methods being the more promising ones. In this report, filamentous fungi isolated from Portuguese grapes were assessed for ochratoxin A degradation capabilities. It was observed that 51 of the 76 tested strains, predominantly aspergillus species, were able to degrade more than 80% of ochratoxin A added to the culture medium and that the most potent species (more than 95% of initial amount) were the black aspergilli, A. clavatus, A. ochraceus, A. versicolor, and A. wentii. Other fungi frequently isolated from grapes, such as Alternaria, Botrytis, Cladosporium, and Penicillium, also showed significant degradation capabilities. It was observed that the compounds obtained from the degradation of ochratoxin A by black aspergilli and by A. ochraceus and A. wentii strains were different.     

Effects of ionic strength and nature of the cation on the desorption of fluoride from soil

The reaction between soil and added fluoride was accelerated by incubating at a high temperature. Desorption of the fluoride was then studied using solutions of chloride salts of several cations at a range of solution : soil ratios and for periods which ranged from 1 h to 4 days. Fluoride desorbed was related to the experimental variables by a regression equation. When the solution : soil ratio was small and hence only small amounts of fluoride were desorbed, decreasing the concentration of salts increased the concentration of fluoride in the solution. The concentration in the solution was lower for calcium chloride than for sodium or potassium chloride. Amongst the monovalent cations, the concentration of fluoride was highest for salts of the cations of lowest atomic number. Thus the greater the average distance between the charge conveyed to the surface by the adsorbed fluoride and the cation which balanced it, the higher the fluoride concentration in the solution. As the solution : soil ratio was increased, the differences between the cations in their effects on fluoride desorption decreased and seemed to disappear as the solution : soil ratio became very large. This contrasts with previously observed effects on phosphate. It is suggested that the difference may have arisen because appreciable desorption of fluoride occurs by exchange with hydroxyl ions rather than by escape of the fluoride ion together with its counter ion.     

Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products

Penicillium expansum is known for its destructive rot and patulin production in apple juice. According to the literature, P. expansum can, among other compounds, produce citrinin, ochratoxin A, patulin, penitrem A, and rubratoxin B. In this study the qualitative production of metabolites was examined using TLC (260 isolates), HPLC (85 isolates), and MS (22 isolates). The results showed that none of the 260 isolates produced ochratoxin A, penitrem A, or rubratoxin B. However, chaetoglobosin A and communesin B were produced consistently by all 260 isolates. Patulin and roquefortine C were produced by 98% of the isolates. Expansolides A/B and citrinin were detected in 91 and 85% of the isolates, respectively. Chaetoglobosins and communesins were detected in naturally infected juices and potato pulp, whereas neither patulin nor citrinin was found. Because most P. expansum isolates produce patulin, citrinin, chaetoglobosins, communesins, roquefortine C, and expansolides A and B, foods contaminated with this fungus should ideally be examined for chaetoglobosin A as well as patulin.     

Highly sensitive analysis of iodide anion in seaweed as pentafluorophenoxyethyl derivative by capillary gas chromatography

A simple and sensitive gas chromatography (GC) method is described for the trace analysis of iodide anion (iodide) in processed seaweed as an organic derivative. The method is based on the derivatization of aqueous iodide extracted from seaweed with 2-(pentafluorophenoxy)ethyl 2-(piperidino)ethanesulfonate in toluene using tetra-n-hexylammonium bromide as a phase-transfer catalyst. The resulting pentafluorophenoxyethyl iodide is highly responsive to an electron-capture detector (ECD) and was analyzed by GC-ECD, giving a low detection limit of approximately 2.7 nM (2.7 fmol/microL injected). Interferences of some common anions in the analysis of iodide were studied and proved to be minimal. Application of the method to the analysis of iodide in processed seaweed was performed.     

不溶性钾矿产品部分替代氯化钾下晚稻钾素吸收特征

在南方稻区通过大田试验研究不溶性钾矿产品—颗粒硅钙钾肥和多元素微孔矿物肥部分替代氯化钾对晚稻产量、经济效益以及对钾素吸收利用的影响。结果表明:施钾使晚稻增产200.4~577.4 kg/hm~2,40%颗粒硅钙钾肥替代氯化钾增产122.8 kg/hm~2,其他不溶性钾矿产品(颗粒硅钙钾肥、微孔矿物肥)替代氯化钾处理使晚稻减产191.5~254.2 kg/hm~2。施氯化钾和40%颗粒硅钙钾肥使晚稻增收574.7和465.7元/hm~2,不溶性钾矿产品部分替代氯化钾均降低晚稻产投比。施钾处理均能够提高晚稻累积吸钾量和吸钾总量。不溶性钾矿产品部分替代氯化钾对晚稻累积吸钾量和吸钾总量效果不明显,对晚稻钾素利用率的提高没有明显效果。南方稻区晚稻生产中不溶性钾矿产品可以部分替代氯化钾,但不能给当季晚稻生产带来良好的经济效益。     

Fumonisin B2 production by Aspergillus niger

The carcinogenic mycotoxin fumonisin B2 was detected for the first time in the industrially important Aspergillus niger. Fumonisin B2, known from Fusarium verticillioides and other Fusaria, was detected in cultures of three full genome sequenced strains of A. niger, in the ex type culture and in a culture of F. verticillioides by electrospray LC-MS analysis of methanolic extracts from agar plugs of cultures grown on several substrates. Whereas F. verticillioides produced fumonisins B1, B2, and B3 on agar media based on plant extracts, such as barley malt, oat, rice, potatoes, and carrots, A. niger produced fumonisin B2 best on agar media with a low water activity, including Czapek yeast autolysate agar with 5% NaCl. Of the media tested, only rice corn steep agar supported fumonisin production by both F. verticillioides and A. niger. However, A. niger had a different regulation of fumonisin production and a different quantitative profile of fumonisins, producing only B2 as compared to F. verticillioides. Fumonisin production by A. niger, which is a widely occurring species and an extremely important industrial organism, will have very important implications for biotechnology and especially food safety. A. niger is used for the production of citric acid and as producer of extracellular enzymes, and also as a transformation host for the expression of heterologous proteins. Certain strains of A. niger produce both ochratoxin A and fumonisins, so some foods and feeds may potentially contain two types of carcinogenic mycotoxins from this species.     

On-line generation of cyanogen chloride in semiautomated determination of niacin and niacinamide in food products

The current AOAC procedure for semiautomated determination of niacin specifies the use of externally generated cyanogen bromide. Because of the safety concerns in handling this material, we investigated the use of an alternative system of generating cyanogen chloride in situ, using chloramine-T and potassium cyanide. Recovery studies conducted on 9 different food products yielded average recoveries of 101%. A repeatability study resulted in a measured coefficient of variation of 2.9%. The AOAC niacin method was compared with this semiautomated method; 115 paired analyses on 8 different food types over 6 separate analytical replications indicated no significant difference by a paired t-test at the 95% confidence level.     

Liquid chromatographic determination of ochratoxin A in coffee beans and coffee products

A liquid chromatographic method for the determination of ochratoxin A in coffee beans (green and roast), instant coffee, and coffee drink is described. The sample is subjected to extraction with methanol-1% aqueous sodium bicarbonate (1 + 1) and C18 cartridge cleanup. The extract is chromatographed on a Nucleosil 5C18 column with a mobile solvent of acetonitrile-water-0.2M phosphate buffer pH 7.5 (50 + 47 + 3) containing 3 mM cetyltrimethylammonium bromide as an ion-pair reagent. Ochratoxin A is detected with a fluorometer (excitation 365 nm, emission 450 nm). The sensitivity was increased 20-fold by using ion-pair resolution. The detection limits corresponded to 2 micrograms/kg for coffee beans, 5 micrograms/kg for instant coffee, and 0.2 microgram/kg for coffee drink. The recoveries from coffee products were generally better than 80.7% and the relative standard deviations were 3.43-5.93%. The peak coinciding with ochratoxin A can be confirmed by treatment using alcohol (methanol, ethanol, or n-propanol) and H2SO4.     

Determination of ochratoxin A by monoclonal antibody-based enzyme immunoassay

A simple and rapid indirect enzyme-linked immunosorbent assay was developed for the quantitative determination of ochratoxin A in barley after the successful production of a high affinity, specific monoclonal antibody. A rapid sample cleanup was achieved by extracting ochratoxin A from barley with chloroform and partitioning the toxin into bicarbonate buffer; the buffer solution was then added directly to the assay plate and ochratoxin A content was assessed. Recoveries were greater than 85% and detection limits were 5 micrograms ochratoxin A/kg barley.     

Mycotoxins in grain dust: method for analysis of aflatoxins, ochratoxin A, zearalenone, vomitoxin, and secalonic acid D

A multimycotoxin method is presented to quantitate aflatoxins, ochratoxin A, zearalenone, secalonic acid D, and vomitoxin in grain dust. Dust spiked with these mycotoxins was extracted sequentially with methylene chloride followed by acetonitrile-water (86 + 14). Vomitoxin was recovered in the latter extract and all other mycotoxins were recovered in the methylene chloride. Aflatoxins and ochratoxin were quantitated by fluorescence measurement on silica thin layer chromatographic plates. The other mycotoxins were quantitated after cleanup by reverse phase liquid chromatography and ultraviolet detection. Recoveries from dust spiked in the parts per billion (ng/g) range were approximately 80% (SD = 15-29%) for all mycotoxins. Minimum detectable amounts ranged from less than 0.5 ng/g for aflatoxins to 20 ng/g for zearalenone.     

Fungal microflora and ochratoxin a risk in French vineyards

To evaluate the ochratoxin A risk in French vineyards, five winemaking regions were investigated. An exhaustive survey of the fungal microflora of 60 grape samples was carried out at two development stages of the berries: end of veraison and harvest time. Potentially toxinogenic fungi isolated from grapes were assessed in vitro for ochratoxin A production. Ochratoxin A was also quantified in musts by high-performance liquid chromatography after cleanup on immunoaffinity columns. Among the 90 species identified, almost half are listed as mycotoxin producers, but only 2 are potentially ochratoxinogenic: Aspergillus carbonarius and Aspergillus niger. Among these strains, only A. carbonarius, isolated from the Languedoc region at harvest time, was found to produce ochratoxin A. These results were in accordance with the presence of ochratoxin A in French southern region musts (0.01-0.43 microg/L) and confirmed the major implication of A. carbonarius in ochratoxin A contamination.     

Liquid chromatographic determination of aflatoxins, ochratoxin A, and zearalenone in grains, oilseeds, and animal feeds by post-column derivatization and on-line sample cleanup

A liquid chromatographic method using on-line sample cleanup, reverse flow analytical column loading, gradient elution, and postcolumn derivatization with iodine permits direct, rapid determination of aflatoxins B1, B2, G1, and G2, as well as ochratoxin A and zearalenone. Limits of quantitation are 5 ppb for the aflatoxins and ochratoxin A and 30 ppb for zearalenone. This procedure performs well as a multimycotoxin screen for cereal grains and oilseeds, with more limited success in complete animal feeds.     

Rapid microchemical identification of four phenothiazine antiemetics with gold bromide and iodine-potassium iodide reagents: collaborative study

A microchemical method was developed for the rapid identification of 4 phenothiazine antiemetics. Perphenazine, promethazine, thiethylperazine, and triflupromazine were positively identified and differentiated with the aid of a gold bromide reagent and an iodine-potassium iodide reagent. Only promethazine and triflupromazine yielded microcrystalline derivatives with gold bromide; only perphenazine and thiethylperazine reacted with iodine-potassium iodide. For each pair of positive reactions, the crystalline products were morphologically distinguishable under a microscope. The 2 tests were collaboratively studied by 7 independent laboratories and found to be simple, rapid, and effective for identifying the phenothiazines of interest. The method has been adopted official first action.     

Effects of chronic ingestion of ochratoxin a on blood levels and excretion of the mycotoxin in sheep

Ruminants are relatively resistant to the acutely toxic effects of ochratoxin A, due to extensive degradation of ochratoxin A to its less toxic metabolite ochratoxin alpha by rumen microorganisms. However, most estimates of the degradation capacity for ochratoxin A in ruminants are based on in vitro studies. In the current study, the metabolism of ochratoxin A was investigated over a period of 29 days, feeding various doses of the mycotoxin (0, 9.5, 19.0, and 28.5 mug ochratoxin A/kg body weight) to sheep. Animals were fed diets consisting of 70% concentrates and 30% grass silage. Significant concentrations of undegraded ochratoxin A were detected in serum of sheep at all levels of ochratoxin A tested. Serum concentrations of ochratoxin A slightly accumulated with time of exposure and were linearly dependent on the administered dose of ochratoxin A. Furthermore, a constant proportion (6-8%) of the dose was excreted in the urine. The results of this study indicate that even at moderate to low levels of ochratoxin A in the diet, considerable amounts of the mycotoxin are absorbed by ruminants and may accumulate in tissues. Therefore, feeding of ochratoxin A-contaminated feedstuffs to ruminants does not seem to be a reliable means for using these feedstuffs.     

Ultraviolet-C and induced stilbenes control ochratoxigenic Aspergillus in grapes

This study investigated the efficacy of ultraviolet-C (254 nm) and induced stilbenes to inhibit Aspergillus carbonarius and Aspergillus tubingensis and control ochratoxin A production in grapes. In addition, the stilbene synthesis as a response to UV-C treatment and to infection of ochratoxigenic Aspergillus was compared. The initial microbial inactivation by a previously optimized UV-C illumination protocol for increasing trans-resveratrol content in grapes (50 W/m (2), 40 cm, 60 s) was similar on undamaged and damaged grapes, achieving 1.2 and 1.3 log conidia/100 g reductions, respectively. After 5 days of storage at 22 degrees C, UV-C treatment and the stilbenes induced by UV-C inhibited ochratoxigenic Aspergillus growth in undamaged grapes. UV-C elicited the biosynthesis of trans-resveratrol, while microbial infection and tissue damage triggered the biosynthesis of trans-piceid. trans-Resveratrol was not synthesized as a consequence of ochratoxigenic Aspergillus contamination. However, when trans-resveratrol was synthesized by UV-C, it contributed to inhibiting the development of ochratoxin A producing aspergilli. Furthermore, UV-C treatment also contributed to decrease ochratoxin A production by ochratoxigenic aspergilli. Therefore, UV-C is a promising emerging technology either for reducing the potential ochratoxigenic risk in grapes, which is of particular interest to the wine industry, and also for increasing trans-resveratrol content of grapes, which would provide an added value to the wine.     

Enzyme-linked immunosorbent assay of ochratoxin A in barley

A noncompetitive, double antibody enzyme-linked immunosorbent assay for ochratoxin A using microtitration plates has been developed and applied to samples of barley. The anti-ochratoxin A antiserum, which is used at high dilution, does not cross-react significantly with ochratoxin B or ochratoxin a. Assay sensitivity for determination of the toxin in barley samples is 60 ng/kg. Minimal sample preparation is required before assay.     

A spectrophotometric procedure, using carboxypeptidase A, for the quantitative measurement of ochratoxin A.

In the method described, ochratoxin A is eleaved into ochratoxin alpha (free isocoumarin chromophore) and phenylaline, using carboxypeptidase. Detection is based on the difference in fluorescence excitation spectra of ochratoxin A (380 NM, maximum) and ochratoxin alpha (340 nm, maximum). The quantitation of ochratoxin A is based on the loss of fluorescence intensity at 380 nm. The method has been used for the quantitative determination of as little as 4 mug ochratoxin A/kg barley and barley meal but it could be extended to other products.     

控释钾肥对大蒜-棉花套作体系产量和土壤钾素供应的影响

于2013-2015年在山东农业大学试验站进行连续3年5季的池栽试验,以硫酸钾基施(CK1)和氯化钾基施(CK2)为对照,探究氯化钾50%基施+50%盛花期追施(KCl D)、氯化钾配施硫磺基施(KCl S)、控释氯化钾基施(CRK)和控释氯化钾配施硫磺基施(CRKS)对棉花各生育期叶片光合特性和土壤速效钾含量变化以及蒜棉套作体系产量的影响,为棉花合理施用钾肥提供理论依据。结果表明,控释氯化钾在土壤中养分释放能够满足棉花各生育期钾素需求。CRKS较普通钾肥基施处理提高了铃期和始絮期土壤速效钾含量,提高了铃期后叶片SPAD值和净光合速率,增加了成铃数和单铃重,皮棉显著增产16.9%~30.9%。CRKS较KCl D和CRK皮棉分别增产12.2%~16.1%和8.7%~10.4%。大蒜蒜薹和鳞茎产量均以CRKS最高,较其余处理分别增产2.8%~27.9%和4.8%~23.5%。CRKS较CK2显著提高了纤维长度、整齐度指数和伸长率。经过3年施肥后,CRKS较普通钾肥基施显著提高了土壤水溶性钾和非特殊吸附钾含量。因此,控释氯化钾配施硫磺在棉花上一次基施代替硫酸钾和氯化钾提高了棉花生长后期土壤钾素供应和有效性,改善了叶片光学特性,提高了棉花产量和品质。