Adsorption of Zearalenone by beta-D-glucans in the Saccharomyces cerevisiae cell wall

Cell walls of yeasts and bacteria are able to complex with mycotoxins and limit their bioavailability in the digestive tract when these yeasts and bacteria are given as feed additives to animals. To identify the component(s) of the yeast cell wall and the chemical interaction(s) involved in complex formation with zearalenone, four strains of Saccharomyces cerevisiae differing in their cell wall glucan and mannan content were tested. Laboratory strains wt292, fks1, and mnn9 were compared with industrial S. cerevisiae strain sc1026. The complex-forming capacity of the yeast cell walls was determined in vitro by modelling the plots of amount of toxin bound versus amount of toxin added using Hill's model. A cooperative relationship between toxin and adsorbent was shown, and a correlation between the amount of beta-D-glucans in cell walls and complex-forming efficacy was revealed (R2 = 0.889). Cell walls of strains wt292 and mnn9, which have higher levels of beta-D-glucans, were able to complex larger amounts of zearalenone, with higher association constants and higher affinity rates than those of the fks1 and sc1026 strains. The high chitin content in strains mnn9 and fks1 increased the alkali insolubility of beta-D-glucans from isolated cell walls and decreased the flexibility of these cell walls, which restricted access of zearalenone to the chemical sites of the beta-D-glucans involved in complex formation. The strains with high chitin content thus had a lower complex-forming capacity than expected based on their beta-D-glucans content. Cooperativity and the three-dimensional structure of beta-D-glucans indicate that weak noncovalent bonds are involved in the complex-forming mechanisms associated with zearalenone. The chemical interactions between beta-D-glucans and zearalenone are therefore more of an adsorption type than a binding type.     

Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae

Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. Analysis of fks1 delta mutants showed a partial K1 killer toxin-resistant phenotype and a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1 delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. Overexpression of FKS2 suppressed the killer toxin phenotype of fks1 delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1 delta cells. In addition, eight out of 12 fks1ts fks2 delta mutants had altered beta-glucan levels at the permissive temperature: the partial killer resistant FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive allele FKS1T605I M761T increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. We analysed Fks1p and Fks2p in beta-1,6-glucan deficient mutants and found that they were mislocalized and that the mutants had reduced in vitro glucan synthase activity, possibly contributing to the observed beta-1,6-glucan defects.     

In situ localization of beta-glucans in the cell wall of Schizosaccharomyces pombe

The chemical composition of the cell wall of Sz. pombe is known as beta-1,3-glucan, beta-1,6-glucan, alpha-1,3-glucan and alpha-galactomannan; however, the three-dimensional interactions of those macromolecules have not yet been clarified. Transmission electron microscopy reveals a three-layered structure: the outer layer is electron-dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense. In intact cells of Sz. pombe, the high-resolution scanning electron microscope reveals a surface completely filled with alpha-galactomannan particles. To better understand the organization of the cell wall and to complement our previous studies, we set out to locate the three different types of beta-glucan by immuno-electron microscopy. Our results suggest that the less dense layer of the cell wall contains mainly beta-1,6-branched beta-1,3-glucan. Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the alpha-galactomannan layer, suggesting that some of the radial filaments contain beta-1,6-branched beta-1,3-glucan. beta-1,6-glucan is preferentially located underneath the alpha-galactomannan layer. Linear beta-1,3-glucan is exclusively located in the primary septum of dividing cells. beta-1,6-glucan only labels the secondary septum and does not co-localize with linear beta-1,3-glucan, while beta-1,6-branched beta-1,3-glucan is present in both septa. Linear beta-1,3-glucan is present from early stages of septum formation and persists until the septum is completely formed; then just before cell division the label disappears. From these results we suggest that linear beta-1,3-glucan is involved in septum formation and perhaps the separation of the two daughter cells. In addition, we frequently found beta-1,6-glucan label on the Golgi apparatus, on small vesicles and underneath the cell membrane. These results give fresh evidence for the hypothesis that beta-1,6-glucan is synthesized in the endoplasmic reticulum-Golgi system and exported to the cell membrane.     

拮抗酵母菌控制玉米赤霉烯酮的研究进展

玉米赤霉烯酮是一种有毒的真菌次生代谢产物,是世界上污染粮食范围最广泛的真菌毒素之一,它不仅具有生殖毒性,还具有细胞毒性、免疫毒性以及致癌作用。传统控制玉米赤霉烯酮的方法主要有物理法和化学法,但上述两种方法都具有局限性,近年来,采用拮抗酵母菌控制谷物中玉米赤霉烯酮展现出良好的应用前景。本文对拮抗酵母菌控制玉米赤霉烯酮的机制研究进行了综述,包括酵母菌细胞壁对玉米赤霉烯酮的吸附作用,酵母菌对玉米赤霉烯酮的降解作用,进一步探讨了相关作用对粮食污染生防效果的影响。     

Induction of TNF-alpha production from human peripheral blood monocytes with beta-1,3-glucan oligomer prepared from laminarin with beta-1,3-glucanase from Bacillus clausii NM-1

We prepared a beta-1,3-glucan oligomer (DP> or = 4) from laminarin (DP: 25-30) derived from Laminaria digitata with beta-1,3-glucanase, and examined its effect on human peripheral blood monocytes. Conditioned medium prepared by incubating monocytes (MC-CM) with the beta-1,3-glucan oligomer showed strong inhibitory activity against the proliferation of human leukemic U937 cells. Since the beta-1,3-glucan oligomer had no direct cytotoxic effect on U937 cells up to 1000 microg/ml, the cytotoxicity of the MC-CM may be due to cytotoxic cytokines produced from monocytes stimulated by the beta-1,3-glucan oligomer. On the other hand, the MC-CM prepared with original laminarin had little effect on the growth of U937 cells. The cytotoxicity of the MC-CM prepared with the beta-1,3-glucan oligomer was significantly reduced by an anti-TNF-alpha antibody, but the anti-TNF-beta antibody had no effect. Our results suggest that the enzymatically depolymerized beta-1,3-glucan oligomer induces TNF-alpha production from human monocytes.     

Toxin production by Fusarium species and occurrence of the toxins in agricultural products]

In rainy autumnal weather and unfavourable storage conditions, certain agricultural products may be severely infested with Fusarium species also in Hungary. Since 1970 the authors have detected more than 200 Fusarium infestations in maize, wheat and mixed fodder. The Fusarium toxins were detected thin-layer chromatographically after extraction with chloroform and purification by means of solumn chromatography. Apart from F-2 (zearalenone) which is the most frequent toxin, other ultraviolet active toxic compounds were disclosed. Of the latter, the compounds F-3 and F-4 were isolated; their infra-red spectra were determined. From the results obtained, it may be concluded that these compounds have structures which resemble that of zearalenone and that they produce toxic effects.     

Mycotoxin contamination of cereal grain commodities in relation to climate in North West Europe

This study aimed to investigate mycotoxin contamination of cereal grain commodities for feed and food production in North Western Europe during the last two decades, including trends over time and co-occurrence between toxins, and to assess possible effects of climate on the presence of mycotoxins. For these aims, analytical results related to mycotoxin contamination of cereal grain commodities, collected in the course of national monitoring programmes in Finland, Sweden, Norway and the Netherlands during a 20-year period, were gathered. Historical observational weather data, including daily relative humidity, rainfall and temperature, were obtained from each of these four countries. In total 6382 records, referring to individual sample results for mycotoxin concentrations (one or more toxins) in cereal grains were available. Most records referred to wheat, barley, maize and oats. The most frequently analysed mycotoxins were deoxynivalenol, 3-acetyl-deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin and zearalenone. Deoxynivalenol had the highest overall incidence of 46%, and was mainly found in wheat, maize and oats. Mycotoxins that showed co-occurrence were: deoxynivalenol and 3-acetyl-deoxynivalenol in oats; deoxynivalenol and zearalenone in maize and wheat; and T-2 toxin and HT-2 toxin in oats. The presence of both deoxynivalenol and zearalenone in wheat increased with higher temperatures, relative humidity and rainfall during cultivation, but the presence of nivalenol was negatively associated with most of these climatic factors. The same holds for both nivalenol and deoxynivalenol in oats. This implies that climatic conditions that are conducive for one toxin may have a decreasing effect on the other. The presence of HT-2 toxin in oats showed a slight decreasing trends over time, but significant trends for other toxins showed an increasing presence during the last two decades. It is therefore useful to continue monitoring of mycotoxins. Obtained results can be used for development of predictive models for presence of mycotoxins in cereal grains.     

桑沟湾养殖牡蛎中贝类毒素监测及预警

利用高效液相色谱-串联质谱(high performance liquid chromatography-tandem mass spectrometry,HPLCMS/MS)方法监测养殖牡蛎中5种腹泻性贝类毒素与6种麻痹性贝类毒素含量变化及分布特征,分别利用HP20大孔型吸附树脂与SP700吸附树脂作为富集树脂,富集养殖海域海水中5种腹泻性贝类毒素与6种麻痹性贝类毒素,利用HPLC-MS/MS检测方法分析其中毒素含量,同步监测了养殖海域内牡蛎与海水中毒素含量,探究了两者之间的关系,建立了牡蛎内贝类毒素含量随海水内贝类毒素含量之间的变化规律。结果显示:在该海域内一共监测到OA、DTX-1、GYM、PTX-2 4种腹泻性贝类毒素与STX、dc STX两种麻痹性贝类毒素;在整个监控期内,海水中所监测贝类毒素随时间变化呈现先增长,达到峰值后逐渐降低趋势。牡蛎中毒素含量与海水中毒素含量呈正相关关系,即牡蛎内毒素的增长随海水内毒素的增长而增长,但牡蛎内毒素含量的峰值出现时间在海水中毒素含量出现峰值之后,延后时间为14 d。根据固相吸附毒素跟踪技术原理,可以提前14 d预警牡蛎内毒素含量。     

Development of a Simultaneous Lateral Flow Strip Test for the Rapid and Simple Detection of Deoxynivalenol and Zearalenone

The objective of this study was to develop a 1‐step simultaneous lateral flow strip test for the rapid and simple detection of deoxynivalenol (DON) and zearalenone (ZEA) in grains. Two monoclonal antibodies (MAbs) against DON and ZEA were respectively conjugated with gold nanoparticles and used to develop a lateral flow strip test for a single toxin and multiple toxins. First, individual lateral flow strips for a single toxin were optimized, and their conditions were used to develop a simultaneous lateral flow strip for multiple toxins. Limits of detection of both lateral flow strip tests for DON and ZEA were the same (DON: 50 ng/mL, ZEA: 1 ng/mL). Both methods showed cross‐reactivity for α‐zearalenol and β‐zearalenol, but no cross‐reaction to other mycotoxins. The results can be completed obtained within 15 min. The cut‐off values of the simultaneous lateral flow strip for the spiked rice and corn were 500 and 10 ng/g for DON and ZEA, respectively. The results demonstrated that the developed simultaneous lateral flow strip test offers a rapid, easy‐to‐use, and portable analytical system and can be used as a convenient qualitative tool for the on‐site detection of DON and ZEA in food and agricultural commodities.     

Naturally occurring Fusarium toxins in New Zealand maize

Twenty samples of maize collected from healthy growing crops and at harvest time and during storage were screened for four Fusarium toxins (deoxynivalenol, diacetoxyscirpenol, T-2 toxin and zearalenone) by gas chromatography-mass spectrometry and thin-layer chromatography. Seventeen samples (85%) contained one or more of these toxins. Zearalenone was present in 15 samples at levels ranging between 0.1 and 16 ppm. Deoxynivalenol, diacetoxyscirpenol and T-2 toxin were found in 11, 6 and 13 samples respectively, all at levels below 1 ppm. This work documents the first reported natural occurrence of Fusarium toxins in New Zealand maize, and it is concluded that all four of the mycotoxins studied are prevalent in apparently healthy standing crops as well as in stored maize.     

Glycogen--a covalently linked component of the cell wall in Saccharomyces cerevisiae.

Glycogen in Saccharomyces cerevisiae is present in two pools, one soluble and intracellular, the other present in the cell wall and rendered water-insoluble owing to its covalent linkage to cell wall beta-glucan. The insoluble glycogen fraction was solubilized using beta-1,3-glucanase. The alpha beta-glucan complex obtained showed intense red staining with iodine and was isolated from free beta-glucans by affinity chromatography using concanavalin A sepharose 4B. Further use of molecular sieving has confirmed that glycogen is linked to beta-glucan as the non-retained fraction on Biogel P2 split into two peaks on treatment with amyloglucosidase. Partial acid hydrolysis and subsequent paper chromatography of the alpha beta-glucan complex isolated revealed the presence of gentiobiose and other higher oligosaccharides, indicating that glycogen is linked to beta-1,3-glucan through a beta-1,6 branch. The insoluble glycogen can be extracted in a soluble form by acetic acid treatment and is known as acid-soluble glycogen. The presence of glycogen in the cell wall is confirmed by controlled enzymatic release of alpha beta-glucan complex using lyticase from Arthobacter luteus without disruption of the plasma membrane, as can be visualized using electron microscopy.     

Natural occurrence of Fusarium toxins in soy food marketed in Germany

A total of 45 samples of soy food including whole beans, roasted soy nuts, flour and flakes, textured soy protein, tofu, proteinisolate including infant formulas and fermented products (soy sauce) were randomly collected in food and health food stores and analysed for Fusarium toxins. A spectrum of 13 trichothecenes of the A-type as well as of the B-type were determined by gas chromatography/mass spectrometry, zearalenone (ZEA), alpha- and beta-zearalenol (alpha- and beta-ZOL) by high performance liquid chromatography (HPLC) with fluorescence and UV-detection. Detection limits ranged between 1 and 19 microg/kg. At least one of the toxins investigated was detected in 11 out of a total of 45 samples of soy food belonging to different commodities. Scirpentriol (SCIRP), 15-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol, T-2 tetraol, HT-2 toxin, deoxynivalenol (DON), 15- and 3-acetyldeoxynivalenol, ZEA, alpha- and beta-ZOL were detected in at least one sample, T-2 triol, T-2, NEO, NIV and FUS-X were not detected in any sample. Five out of 11 samples were positive for one toxin, one sample for two, three, six or seven toxins, two samples for 5 toxins, demonstrating the possibility of a contamination of soy food with a spectrum of Fusarium toxins. SCIRP, DON and ZEA were found up to 108, 260 and 214 microg/kg, the other toxins did not exceed 61 microg/kg. A first insight into the contamination of soy food with a broad spectrum of Fusarium toxins is provided.     

Screening of fermentative bacteria for their ability to bind and biotransform deoxynivalenol, zearalenone and fumonisins in an in vitro simulated corn silage model.

Fermentative bacteria can potentially be utilized to detoxify corn silage contaminated by Fusarium toxins. The objective of the present study was to test a large number of these bacteria for their ability to bind and/or biotransform deoxynivalenol (DON), zearalenone (ZEN) and fumonisins B(1) and B(2) (FB(1), FB(2)) in conditions simulating corn silage. A total of 202 strains were screened in contaminated, pH 4, corn infusion inoculated with 5 x 10(8) CFU ml(-1). Eight Lactobacilli and three Leuconostoc biotransformed ZEN into alpha-zearalenol, but no biotransformation was detected for DON and fumonisins. In contrast, most strains were capable of binding Fusarium toxins. The most effective genera were Streptococcus and Enterococcus, capable of binding up to 33, 49, 24 and 62% of DON, ZEN, FB(1) and FB(2), respectively. The ability to bind Fusarium toxins seems to be a common property of fermentative bacteria and could help to decrease their toxicity in animals.     

Role of surface alteration in determining the mobility of U(VI) in the presence of citrate: implications for extraction of U(VI) from soils

In the present study, the adsorption of U(VI) by a natural iron-rich sand in the presence of citrate was studied over a range of citrate concentrations and pH values. Adsorption of U(VI) on the iron-rich sand decreased in the presence of increasing concentrations of citrate. Adsorption of citrate to the sand was weak under most conditions studied. Several explanations for the adsorption behavior of U(VI) and citrate were investigated, including aqueous complexation of U(VI) by citrate, competition of U(VI) and citrate for adsorption sites, and extraction of Fe and Al from the sorbent surface by citrate (surface alteration). Although aqueous complexation of U(VI) by citrate may still play a significant role, both competitive adsorption and aqueous complexation proved to be inadequate explanations of the adsorption behavior. Both physical surface alteration (i.e., loss of surface area) and chemical surface alteration (i.e., change in the chemical composition of the sand surface) were investigated, with chemical surface alteration controlling the bulk of U(VI) adsorption. Considering these results, remediation schemes that involve organic complexing agents should address the possibility of surface alteration affecting radionuclide adsorption and mobility.     

Mycotoxin production in wheat grains by different Aspergilli in relation to different relative humidities and storage periods

Four different Aspergilli (Aspergillus oryzae, A. parasiticus, A. terreus and A. versicolor) were grown on wheat grains underdifferent degrees of relative humidity 14, 50, 74, 80 and 90%. Samples of wheat grains were taken monthly for a period of six months and examined for mycotoxin production. A. oryzae was found to produce aflatoxins B1, B2, zearalenone, DON and T-2 toxins under elevated degrees of humidity and prolonged periods of storage. A. parasiticus produced aflatoxins B1, G1, NIV, DON and T-2 toxins in high concentrations during a period of not more than three months storage at 14% relative humidity; at an increased level of relative humidity of 74% ochratoxin A, zearalenone and sterigmatocystin were also produced at high levels. The isolate was drastic in toxin production. A. terrus produced toxins at 14% relative humidity (aflatoxin G2 and DON) at levels much higher than any other prevalent degrees of humidity. A. versicolor is highly sensitive to relative humidity and grain moisture content It produced aflatoxins B1, G1, NIV and DON at a relative humidity of 50% and another toxins (aflatoxin G2, ochratoxins A, B and zearalenone) at 74%. The microorganism can be considered a trichothecene producer under suitable relative humidity.     

Evidence of paralytic shellfish poisoning toxin in milkfish in South Taiwan

Natural phytoplankton blooms of the dinoflagellate Alexandrium minutum, milkfish (Chanos chanos) exposed to natural blooms, sediment and mangrove crab (Scylla serrata) were analysed for paralytic shellfish poisoning toxins by high-performance liquid chromatography. The toxin profiles of milkfish and mangrove crab were similar to that of A. minutum collected from blooming fishponds. In a laboratory A. minutum-blooming environment, the stomach and intestine of milkfish accumulated paralytic shellfish poisoning toxins during the exposure period. The non-visceral tissues were non-toxic. However, milkfish lost their entire body burden of toxin on the first day of transferring to a toxic algae-free environment. The result shows that milkfish concentrate paralytic shellfish poisoning toxins in digestive organs and did not retain toxins.     

Mycotoxins in South African traditionally brewed beers

Traditionally brewed alcoholic beverages are regularly consumed by most ethnic black South Africans. Maize and barley, both of which are used for producing locally brewed alcoholic beer, are frequently contaminated by mycotoxin-producing moulds. The study was undertaken to investigate whether these toxins are present in raw grains and the traditional beers imbibed by the local black African population. It was established that the raw ingredients (sorghum, sorghum malt grains, maize grits), commercially produced traditional beers (Utshwala and Utshwala special) and home-brewed beers (Umqombotha, Isiqatha, Imfulamfula) were contaminated by bacteria and fungi (both yeasts and moulds). The contaminating moulds were isolated and identified. The contaminated samples were analysed for aflatoxins B 1 , B 2 , G 1 and G 2 , zearalenone, citrinin, deoxynivalenol, and ochratoxin A using a multi-mycotoxin thin-layer chromatography screening method and the toxins were quantified by high-performance liquid chromatography. Grain samples were infected by Aspergillus flavus , A. alliaceus , A. clavatus , Penicillium spp., Rhizopus spp. and Mucor spp. Sorghum malt grain samples contained the toxin zearalenone. No mycotoxin-producing fungi were present in the fermented beers but two of six commercial beer samples contained aflatoxins (200 and 400 μgl -1 ) and 45% (13 of 29) of the home-brewed beers had zearalenone (range 2.6-426 μg l -1 ) and/or ochratoxin A (3-2340 μg l -1 ).     

Azaspiracid shellfish poisoning: unusual toxin dynamics in shellfish and the increased risk of acute human intoxications

A number of recent acute human intoxications in Europe from the consumption of Irish mussels have been attributed to the presence of a new class of toxins named azaspiracids. The study demonstrates that azaspiracids behave differently from other polyether toxins, and this accounts for most false-negative results in the mouse bioassay employed by regulatory agencies to detect azaspiracids. Typically, polyether toxins are concentrated in the digestive glands of shellfish, but this is not always the situation with azaspiracids. Liquid chromatography-mass spectrometry (LC-MS), especially multiple tandem MS methods, have been applied to demonstrate that azaspiracid (AZA1) and its methyl- and demethyl- analogues, AZA2 and AZA3 respectively, are distributed throughout shellfish tissues. Using conventional mouse bioassay protocols, only 0-40% of the total azaspiracid content of shellfish was used in the assay, which could directly account for false-negative results. It was also observed that the toxin profiles differed significantly in various mussel tissues with AZA1 as the predominant toxin in the digestive glands and AZA3 predominant in the remaining tissues.     

Influence of water activity,temperature and incubation time on the simultaneous production of deoxynivalenol and zearalenone in corn (Zea mays) by Fusarium graminearum

The production of deoxynivalenol and zearalenone by Fusarium graminearum was studied under different culture conditions (water activity, temperature and incubation time). The maximum levels of both toxins were obtained at the 35th day of incubation, the zearalenone level being much higher than the deoxynivalenol. The culture conditions that gave higher yields of deoxynivalenol were at 22 and at 28 °C (6.0 and 5.5 mg/kg ), after 35 days. At an incubation temperature of 28 °C 16 days, followed by 12 °C, for the same time, the production was low (1.1 mg/kg). The highest level of zearalenone was obtained at 28 °C for 16 days, followed by incubation at 12 °C (36.7mg/kg) at the 35th day. When the temperature was constant at 28 °C, the zearalenone production was lower (3.0 mg/kg) than when incubated at 22 °C (12.3 mg/kg), at the 35th day. Fusarium graminearum did not produce deoxynivalenol and zearalenone at 37 °C.     

Food safety implications of the distribution of azaspiracids in the tissue compartments of scallops ( Pecten maximus )

Azaspiracids, a new class of shellfish toxins, have been implicated in several recent incidents of human intoxications following the consumption of mussels ( Mytilus edulis ). A study was undertaken to examine the distribution of azaspiracid poisoning (AZP) toxins in scallops ( Pecten maximus ) and individual shellfish were dissected into five tissue fractions for the determination of toxin composition. Separation of the predominant azaspiracids, AZA1-3, was achieved using reversed-phase liquid chromatography with detection by positive electrospray multiple tandem mass spectrometry. The AZP toxin composition was determined in the adductor muscle (meat), gonad (roe), hepatopancreas (digestive glands), mantle and gill of scallops. Substantial differences in the AZP toxin levels between tissue compartments were observed and toxins were concentrated predominantly, about 85%, in the hepatopancreas. There was also a significant variation in the total toxin levels between individual scallops from the same sample batch and the RSD was 60% (n = 9). Interestingly, although all three AZP toxins were present in phytoplankton and mussels, AZA3 was not detected in the scallop samples examined. It was concluded that to improve food safety, only the adductor muscle and gonad of scallops should be permitted for sale to the public.