Aflatoxin B1 and Sterigmatocystin Binding Potential of Non-Lactobacillus LAB Strains.

Research on the ability of lactic acid bacteria (LAB) to bind aflatoxin B1 (AFB1) has mostly been focusing on lactobacilli and bifidobacteria. In this study, the AFB1 binding capacities of 20 Enterococcus strains belonging to E. casseliflavus, E. faecalis, E. faecium, E. hirae, E. lactis, and E. mundtii, 24 Pediococcus strains belonging to species P. acidilactici, P. lolii, P. pentosaceus, and P. stilesii, one strain of Lactococcus formosensis and L.garviae, and 3 strains of Weissella soli were investigated in MRS broth at 37 °C at 0.2 µg/mL mycotoxin concentration. According to our results, among non-lactobacilli LAB, the genera with the best AFB1 binding abilities were genus Pediococcus, with a maximum binding percentage of 7.6% by P. acidilactici OR83, followed by genus Lactococcus. For AFB1 bio-detoxification purposes, beside lactobacilli, pediococci can also be chosen, but it is important to select a strain with better binding properties than the average value of its genus. Five Pediococcus strains have been selected to compare their sterigmatocystin (ST) binding abilities to AFB1 binding, and a 2-3-fold difference was obtained similar to previous findings for lactobacilli. The best strain was P. acidilactici OR83 with 18% ST binding capacity. This is the first report on ST binding capabilities of non-Lactobacillus LAB strains.

Chemical Constituents from Endophytic Fungus Annulohypoxylon cf. stygium in Leaves of Anoectochilus roxburghii.

The chemical investigation on endophytic fungus Annulohypoxylon cf. stygium in leaves of Anoectochilus roxburghii (Wall.) Lindl. has been performed. Sixteen compounds were isolated and their structures were identified as (-)-notoamide A, (-)-notoamide B, (+)-versicolamide B, notoamide C, notoamide D, stephacidin A, sterigmatocystin, dihydrosterigmatocystin, secosterigmatocystin, versiconol, averufanin, kipukasin D, kipukasin E, diorcinal, palmarumycin CP2 and (-)-(3R)-mellein methyl ether, respectively, by spectroscopic analysis and comparison with literature data. All the compounds were isolated from Annulohypoxylon genus for the first time. Sterigmatocystin and palmarumycin CP2 showed selective cytotoxic activities against HepG2, HeLa, MCF-7 and HT-29.

Isolation and Identification of Three New Sterigmatocystin Derivatives from the Fungus Aspergillus versicolor Guided by Molecular Networking Approach.

Molecular networking approach was applied for the targeted isolation of new sterigmatocystin derivatives, sterigmatocystins A-C, from the marine sponge-derived fungus Aspergillus versicolor. Sterigmatocystin A features a rare 6/6/6/6/5 polycyclic system. The structures of sterigmatocystins A-C, including absolute configurations, were determined on the basis of spectroscopic data and ECD calculations. Sterigmatocystin A showed more stronger promoting angiogenesis activity than the positive control at 1.25 µM level in transgenic fluorescent zebrafish. Sterigmatocystins A-C also exhibited moderate antiviral activity by the inhibition of HSV-2.

Selective recognition and enrichment of sterigmatocystin in wheat by thermo-responsive imprinted polymer based on magnetic halloysite nanotubes.

Two thermo-responsive molecularly imprinted polymers (MHNTs@MIP and MCNTs@MIP) for the selective extraction of sterigmatocystin have been prepared on the surface of the magnetic halloysite nanotubes (MHNTs) and magnetic carbon nanotubes (MCNTs), respectively. 1, 8-dihydroxyanthraquinone, n-isopropyl acrylamide, methacrylic acid, ethylene dimethacrylate and dimethyl sulfoxide were used as the dummy template, thermo-sensitive functional monomer, co-monomer, cross-linker and porogen, respectively. The magnetic properties, adsorption properties as well as the temperature responsive behaviors of MHNTs@MIP and MCNTs@MIP were systematically studied and compared for the first time. Enough saturation magnetizations of MHNTs@MIP (9.42 emu/g) and MCNTs@MIP (10.54 emu/g) were obtained. MHNTs@MIP and MCNTs@MIP also showed controllable adsorption and release behaviors to sterigmatocystin in response to the temperature change (35 °C and 20 °C). Compared with MCNTs@MIP, MHNTs@MIP had higher adsorption affinity (KL = 0.120 L/mg), higher adsorption kinetic (K2 = 0.0100 g/(mg•min)) and higher imprinting factor (5.22) to sterigmatocystin. These results indicated that MHNTs@MIP was favorable adsorbent for the selective separation of sterigmatocystin. Furthermore, the elution conditions of MHNTs@MIP were optimized by response surface methodology. Under the optimal conditions, MHNTs@MIP coupled with high performance liquid chromatography were successfully applied to the selective recognition, purification, enrichment and detection of sterigmatocystin in wheat samples. The recoveries were calculated from 88.62% to 102.9% with RSDs less than 3.5 % and limit of detection of 1.1 µg/kg. This work provided a suitable carrier for the preparation of imprinted polymers and a practical approach for highly selective recognition and determination of analytes in real samples.

Occurrence of the Toxin-Producing Aspergillus versicolor Tiraboschi in Residential Buildings.

In an area representative of a moderate climate zone (Lubuskie Province in Poland), mycological tests in over 270 flats demonstrated the occurrence of 82 species of moulds. Aspergillus versicolor Tiraboschi was often encountered on building partitions (frequency 4: frequently). The ability to synthesize the carcinogenic sterigmatocystin (ST) means that it poses a risk to humans and animals. Biotoxicological tests of biomasses of A. versicolor were conducted in the Microbiological and Toxicological Laboratory, using the planarians Dugesia tigrina (Girard). The obtained results of the tests covered a broad range of toxicity levels of isolated strains: from weakly toxic (100-1000 mg·L(-3)) to potently toxic (1-10 mg·L(-3)). The high-performance liquid chromatography (HPLC) physicochemical method confirmed the ability of A. versicolor strains to synthesize sterigmatocystin. All of the samples of the air-dry biomasses of the fungi contained ST in the range between 0.03 and 534.38 mg·kg(-1). In the bio-safety level (BSL) classification A. versicolor belongs to category 1. Additionally, A. versicolor is an allergenic mould.

The capability of fungi isolated from moldy dwellings to produce toxins.

OBJECTIVES: The main objective was analysis and assessment of toxinogenic capabilities of fungi isolated from moldy surfaces in residential rooms in an urban agglomeration situated far from flooded areas in moderate climate zone. MATERIAL AND METHODS: The assessment of environmental exposure to mycotoxins was carried out in samples collected from moldy surfaces in form of scrapings and airborne dust from 22 moldy dwellings in winter season. In each sample 2 mycotoxins were analyzed: sterigmatocystin and roquefortine C produced by Aspergillus versicolor and Penicillium chrysogenum, respectively. Mycotoxins were analyzed by high-performance liquid chromatography (HPLC) in: scrapings from moldy surfaces, mixture of all species of fungi cultured from scrapings on microbiological medium (malt extract agar), pure cultures of Aspergillus versicolor and Penicillium chrysogenum cultured from scrapings on microbiological medium; mycotoxins in the indoor air dust were also analyzed. RESULTS: The production of sterigmatocystin by individual strains of Aspergillus versicolor cultured on medium was confirmed for 8 of 13 isolated strains ranging 2.1-235.9 µg/g and production of roquefortine C by Penicillium chrysogenum for 4 of 10 strains ranging 12.9-27.6 µg/g. In 11 of 13 samples of the mixture of fungi cultured from scrapings, in which Aspergillus versicolor was found, sterigmatocystin production was at the level of 3.1-1683.2 µg/g, whereas in 3 of 10 samples in which Penicillium chrysogenum occurred, the production of roquefortine C was 0.9-618.9 µg/g. The analysis did not show in any of the tested air dust and scrapings samples the presence of analyzed mycotoxins in the amount exceeding the determination limit. CONCLUSIONS: The capability of synthesis of sterigmatocystin by Aspergillus versicolor and roquefortine C by Penicillium chrysogenum growing in mixtures of fungi from scrapings and pure cultures in laboratory conditions was confirmed. The absence of mycotoxins in scrapings and air dust samples indicates an insignificant inhalatory exposure to mycotoxins among inhabitants in moldy flats of urban agglomeration situated far from flooded territories. Int J Occup Med Environ Health 2016;29(5):823-836.

Analysis of sterigmatocystin in cereals, animal feed, seeds, beer and cheese by immunoaffinity column clean-up and HPLC and LC-MS/MS quantification.

A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 µg kg(-1) to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68% to 106%, with repeatability from 4.2% to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 µg kg(-1). For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 µg kg(-1) the recoveries were 94% and 104%, and precision (RSDs) were 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02 and 0.6 µg kg(-1) respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS.

Determination of sterigmatocystin in feed by LC-MS/MS.

An LC-MS/MS method is proposed for the analysis of sterigmatocystin in cereals and feed. The method is based on a solid-liquid extraction and a dilute-and-shoot approach. Accuracy and precision were established at the LOQ (1 µg kg(-1)); the mean overall recovery (n = 6) was 98%, with a confidence interval of 3.8% and a CV% of 3.7%. Accuracy and precision were also assessed at three other concentration levels (2.03, 5.07 and 10.14 µg kg(-1); six replicates per level). The mean overall recovery (n = 24, LOQ included) was 99% with a confidence interval of 0.8% and a CV% of 1.9%. The method was then applied to 14 naturally incurred feed samples. Aflatoxin B1 was present in the range 28.7-240.1 µg kg(-1), while lower concentrations of sterigmatocystin were found (0.7-2.2 µg kg(-1)). This method may represent a valuable choice, ensuring a high level of accuracy and precision, as well as high-throughput performance. Therefore, it meets the recent EFSA opinion recommendation in terms of availability of fast and sensitive methods (recommended LOQ = 1.5 µg kg(-1)) in order to increase data collection to allow for the assessment of dietary exposure.

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for sterigmatocystin in cereal and oil products.

Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg · mL(-1)) ELISA format, in which the antibody was diluted to 1:80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng · g(-1) for wheat, 0.06 ng · g(-1) for maize, and 0.1 ng · g(-1) for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90-104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products.

Chemical and bioactive natural products from Microthyriaceae sp., an endophytic fungus from a tropical grass.

UNLABELLED: In screening for natural products with antiparasitic activity, an endophytic fungus, strain F2611, isolated from above-ground tissue of the tropical grass Paspalum conjugatum (Poaceae) in Panama, was chosen for bioactive principle elucidation. Cultivation on malt extract agar (MEA) followed by bioassay-guided chromatographic fractionation of the extract led to the isolation of the new polyketide integrasone B (1) and two known mycotoxins, sterigmatocystin (2) and secosterigmatocystin (3). Sterigmatocystin (2) was found to be the main antiparasitic compound in the fermentation extract of this fungus, possessing potent and selective antiparasitic activity against Trypanosoma cruzi, the cause of Chagas disease, with an IC50 value of 0.13 µmol l(-1) . Compounds 2 and 3 showed high cytotoxicity against Vero cells (IC50 of 0.06 and 0.97 µmol l(-1) , respectively). The new natural product integrasone B (1), which was co-purified from the active fractions, constitutes the second report of a natural product possessing an epoxyquinone with a lactone ring and exhibited no significant biological activity. Strain F2611 represents a previously undescribed taxon within the Microthyriaceae (Dothideomycetes, Ascomycota). SIGNIFICANCE AND IMPACT OF THE STUDY: The present study attributes new antiparasitic and psychoactive biological activities to sterigmatocystin (2), and describes the structure elucidation of the new natural product integrasone B (1), which possesses a rare epoxyquinone with a lactone ring moiety. This is also the first report of sterigmatocystin (2) isolation in a fungal strain from this family, broadening the taxonomic range of sterigmatocystin-producing fungi. The study also presents taxonomic analyses indicating that strain F2611 is strongly supported as a member of the Microthyriaceae (Ascomycota), but is not a member of any previously known or sequenced genus.

Three new sterigmatocystin analogues from marine-derived fungus Aspergillus versicolor MF359.

During the systematic screening of active compounds from marine-derived fungi, the extract of a strain of Aspergillus versicolor MF359 isolated from a marine sponge of Hymeniacidon perleve was identified for detailed chemical investigation. Three new secondary metabolites, named hemiacetal sterigmatocystin (1), acyl-hemiacetal sterigmatocystin (2), and 5-methoxydihydrosterigmatocystin (3), together with a known compound, aversin (4), were characterized. 1 represents a first structure of sterigmatocystin hemiacetal from nature. The antibacterial activities of these identified compounds were evaluated against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. Compound 3 showed activity against S. aureus and B. subtilis with MIC values of 12.5 and 3.125 µg/mL, respectively.

[Secondary fungal metabolites (mycotoxins) in lichens of different taxonomic groups].

Secondary fungal metabolites (mycotoxins) in 22 lichen species of the families Parmeliaceae, Nephromataceae, Umbilicariaceae, Ramalinaceae, Cladoniaceae, Peltigeraceae, and Teloschistaceae were identified determined by enzyme immunoassay enzyme-linked immunosorbent assay. The following mycotoxins were identified found in these lichens in a broad concentration range with a frequency of 70-100%: sterigmatocystin (7-2090 ng/g), alternariol (20-6460 ng/g), and emodin (45-94500 ng/g). Mycophenolic acid frequently occurred in 19 lichen species; citrinin, in 17 species; diacetoxyscirpenol, in 11 species; cyclopiazonic acid, in 10 species; and zearalenone, in 9 species. PR toxin was regularly detected in three lichen species; deoxynivalenol, fumonisins, and ochratoxin A, in two species; and T-2 toxin and ergot alkaloids, in one species. Aflatoxin B1 was detected in only six species with a frequency of 2-42%, whereas roridin A was identified present in 10% of Hypogymnia physodes samples.

Sterigmatocystins from the deep-sea-derived fungus Aspergillus versicolor.

Three new sterigmatocystin derivatives, oxisterigmatocystin A (1), oxisterigmatocystin B (2) and oxisterigmatocystin C (3), together with one known compound, 5-methoxysterigmatocystin (4), were isolated from the deep-sea-derived fungus Aspergillus versicolor. The structures of the new compounds were elucidated by spectroscopic methods. The cytotoxicities of compounds 1-4 were evaluated against the A-549 and HL-60 cell lines. Compound 4 exhibited moderate cytotoxicities against the A-549 and HL-60 cell lines with IC(50) value of 3.86 and 5.32 µM, respectively.

Larvicidal activity of metabolites from the endophytic Podospora sp. against the malaria vector Anopheles gambiae.

In a screening for natural products with mosquito larvicidal activities, the endophytic fungus Podospora sp. isolated from the plant Laggera alata (Asteraceae) was conspicuous. Two xanthones, sterigmatocystin (1) and secosterigmatocystin (2), and an anthraquinone derivative (3) 13-hydroxyversicolorin B were isolated after fermentation on M(2) medium. These compounds were characterised using spectroscopic and X-ray analysis and examined against third instar larvae of Anopheles gambiae. The results demonstrated that compound 1 was the most potent one with LC(50) and LC(90) values of 13.3 and 73.5 ppm, respectively. Over 95% mortality was observed at a concentration 100 ppm after 24 h. These results compared farvorably with the commercial larvicide pylarvex® that showed 100% mortality at the same concentration. Compound 3 was less potent and had an LC(50) of 294.5 ppm and over 95% mortality was achieved at a concentration of 1,000 ppm. Secosterigmatocystin (2) revealed relatively weak activity and therefore LC values were not determined.

[The secondary metabolites of the HS-3 Alternaria sp. fungus associated with holothurians].

OBJECTIVE: The metabolites of HS-3 associated with holothurians were studied, which was identified by molecular biology as Alternaria sp.. METHODS: The holothurians were gathered from the Sea of Zhifu Islet, Shandong Province. HS-3 Alternaria sp. was culternitived in potato medium, and four compound was got by TLC, chromatography and HPLC, and 1-hydroxyl-3-methylanthracene-9,10-dione (1), chrysophanol (2), sterigmatocystin (3) and cerebroside (4) were elucated by modern spectrum. CONCLUSION: All of this provides scientific data for further study of holothurians, and the four coumpouns are isolated from the microbe associated with holothurians for the first time.

Cottoquinazoline A and cotteslosins A and B, metabolites from an Australian marine-derived strain of Aspergillus versicolor.

An Australian marine-derived isolate of Aspergillus versicolor (MST-MF495) yielded the known fungal metabolites sterigmatocystin, violaceol I, violaceol II, diorcinol, (-)-cyclopenol, and viridicatol, along with a new alkaloid, cottoquinazoline A (1), and two new cyclopentapeptides, cotteslosins A (2) and B (3). Structures for 1-3 and the known compounds were determined by spectroscopic analysis. The absolute configurations of 1-3 were addressed by chemical degradation and application of the C(3) Marfey's method. The use of "cellophane raft" high-nutrient media as a device for up-regulating secondary metabolite diversity in marine-derived fungi is discussed. The antibacterial properties displayed by A. versicolor (MST-MF495) were attributed to the phenols violaceol I, violaceol II, and diorcinol, while cotteslosins 2 and 3 were identified as weak cytotoxic agents.

Botryolides A-E, decarestrictine analogues from a fungicolous Botryotrichum sp. (NRRL 38180).

Four new decarestrictine analogues (botryolides A-D; 1- 4), a biosynthetically related gamma-lactone (botryolide E; 5), and the known compounds decarestrictine D ( 6) and sterigmatocystin have been isolated from cultures of a fungicolous isolate of Botryotrichum sp. (NRRL 38180). The structures of these compounds were determined by analysis of 2D NMR and ESIMS data. The relative configurations of 1- 5 were established on the basis of NMR data and/or X-ray diffraction analysis, while the absolute configuration of 1 was assigned using the modified Mosher method.

Kipukasins, nucleoside derivatives from Aspergillus versicolor.

Seven new aroyl uridine derivatives (kipukasins A-G; 1-7) were isolated from solid-substrate fermentation cultures of two different Hawaiian isolates of Aspergillus versicolor. The structures of compounds 1-7 were determined by analysis of NMR and MS data. The nucleoside portion of lead compound 1 was assigned as uracil-1-beta-D-ribofuranoside by spectral comparison with an authentic standard. The bioactivity of the original A. versicolor extracts was accounted for mainly by the presence of the known metabolite sterigmatocystin, but kipukasins A and B showed modest activity in assays against Gram-positive bacteria.

[Production and characterization of sterigmatocystin].

Two strains of Aspergillus versicolor producing ST at 550.8 mg.kg-1 substrate and 1160.8 mg.kg-1 substrate were selected to inoculate 4 kg solid ST-producing media. After 35 days stationary incubation at 28 degrees C in the dark, 2271.6 mg of pale-yellow needle-shaped crystals were isolated and purified from the culture with a procedure applying column chromatography and recrystallization method. The crystal was proved to be sterigmatocystin by spectroanalysis and some physico-chemical analysis. The purity of the final material obtained were more that 99.9% as shown by HPLC and TLC detection. With this procedure, ST was obtained at about one tenth of its commercial cost.

Natural occurrence of mycotoxins in different spices in Egypt.

A total of 120 different samples belonging to 24 kinds of species collected from different places at Assiut Governorate (Egypt) were examined for the natural occurrence of mycotoxins. TLC analysis of spice extracts revealed the presence of aflatoxins (8-35 micrograms/kg) in 16 samples of anise, black pepper, caraway, black cumin, fennel, peppermint, coriander and marjoram, sterigmatocystin (10-23 micrograms/kg) in ten samples of red pepper, caraway, cumin and marjoram and citrinin (8-12 micrograms/kg) in two samples of black cumin, while ochratoxin A and zearalenone could not be detected.