Jesper Mølgaard Mogensen

Review of secondary metabolites and mycotoxins from the Aspergillus niger group

AbstractFilamentous fungi in the Aspergillus section Nigri (the black aspergilli) represent some of the most widespread food and feed contaminants known but they are also some of the most important workhorses used by the biotechnological industry. The Nigri section consists of six commonly found species (excluding A. aculeatus and its close relatives) from which currently 145 different secondary metabolites have been isolated and/or detected. From a human and animal safety point of view, the mycotoxins ochratoxin A (from A. carbonarius and less frequently A. niger) and fumonisin B2 (from A. niger) are currently the most problematic compounds. Especially in foods and feeds such as coffee, nuts, dried fruits, and grape-based products where fumonisin-producing fusaria are not a problem, fumonisins pose a risk. Moreover, compounds such as malformins, naptho-γ-pyrones, and bicoumarins (kotanins) call for monitoring in food, feed, and biotechnology products as well as for a better toxicological evaluation, since they are often produced in large amounts by the black aspergilli. For chemical differentiation/identification of the less toxic species the diketopiperazine asperazine can be used as a positive marker since it is consistently produced by A. tubingensis (177 of 177 strains tested) and A. acidus (47 of 47 strains tested) but never by A. niger (140 strains tested). Naptho-γ-pyrones are the compounds produced in the highest quantities and are produced by all six common species in the group (A. niger 134 of 140; A. tubingensis 169 of 177; A. acidus 44 of 47; A. carbonarius 40 of 40, A. brasiliensis 18 of 18; and A. ibericus three of three). FigureImage of Aspergillus niger growing on YES agar, and the resulting extract analysed by LCDAD-TOFMS

Production of Fumonisin B2 and B4 by Aspergillus niger on Grapes and Raisins

The recent discovery of fumonisin production in Aspergillus niger, raises concerns about the presence of these mycotoxins in grapes and raisins as well as other commodities where A. niger is a frequent contaminant. Here we investigate the potential production of fumonisins in A. niger cultured on grapes and raisins. Sixty-six A. niger, 4 A. tubingensis, and 16 A. acidus strains isolated from raisins were tested for fumonisin production on laboratory media. Neither A. tubingensis nor A. acidus strains produced fumonisins, but 77% of A. niger strains did. None of the strains produced ochratoxin A. Ten selected fumonisin producing A. niger strains were further able to produce fumonisin B(2) and fumonisin B(4) on grapes in the range 171-7841 microg fumonisin B(2)/kg and 14-1157 microg fumonisin B(4)/kg. Four selected strains were able to produce fumonisin B(2) (5-6476 microg/kg) and fumonisin B(4) (12-672 microg/kg) on raisins.

Widespread Occurrence of the Mycotoxin Fumonisin B2 in Wine

Fumonisins are important mycotoxins because they are suspected to cause human and animal toxicoses by the consumption of contaminated corn-based food and feeds. However, with the discovery of fumonisin production in grapes by Aspergillus niger , wine may also be a fumonisin-containing commodity. In the present study, we have developed a simple and quantitative cation-exchange-based purification method for the subsequent isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of fumonisins in wine. A comparative study of seven different solid-phase extraction (SPE) columns showed that polymeric mixed-mode reversed-phase (RP) cation-exchange columns were superior to classic silica-based cation and mixed-mode cation-exchange columns. A total of 77 wine samples from 13 countries were subsequently tested, and surprisingly, 18 (23%) were found to contain fumonisin B(2) in the range of 1-25 microg/L. These findings were further confirmed by immunoaffinity purification and re-analysis of the positive cation-exchanged extracts.

Effect of temperature and water activity on the production of fumonisins by Aspergillus niger and different Fusarium species

BackgroundFumonisins are economically important mycotoxins which until recently were considered to originate from only a few Fusarium species. However recently a putative fumonisin gene cluster was discovered in two different Aspergillus niger strains followed by detection of an actual fumonisin B2 (FB2) production in four strains of this biotechnologically important workhorse.ResultsIn the present study, a screening of 5 A. niger strains and 25 assumed fumonisin producing Fusarium strains from 6 species, showed that all 5 A. niger strains produced FB2 and 23 of 25 Fusarium produced fumonisin B1 and other isoforms (fumonisin B2 and B3). Five A. niger and five Fusarium spp. were incubated at six different temperatures from 15-42°C on Czapek Yeast Agar +5% salt or Potato Dextrose Agar. A. niger had the highest production of FB2 at 25-30°C whereas Fusarium spp. had the maximal production of FB1 and FB2 at 20-25°C. Addition of 2.5-5% NaCl, or 10-20% sucrose increased the FB2 production of A. niger, whereas addition of glycerol reduced FB2 production. All three water activity lowering solutes reduced the fumonisin production of the Fusarium species.ConclusionThe present study shows that the regulation of fumonisin production is very different in A. niger and Fusarium, and that food and feeds preserved by addition of sugar or salts may be good substrates for fumonisin B2 production by A. niger.

Aspergillus acidus from Puerh tea and black tea does not produce ochratoxin A and fumonisin B2.

Puerh tea is a unique Chinese fermented tea. Unlike other teas it is stored for a long period of time. Aspergillus niger is claimed to be the dominant microorganism in the Puerh tea manufacturing process and also to be common on tea in general. A. niger sensu stricto is known to produce the mycotoxins ochratoxin A, fumonisins B(2) and B(4). With this in mind, we performed a preliminary study to determine if production of these mycotoxins by black Aspergilli isolated from Puerh and black tea can occur. An examination of 47 isolates from Puerh tea and black tea showed that none of these was A. niger. A part of the calmodulin gene in 17 isolates were sequenced, and these 17 isolates were all identified as Aspergillus acidus (=A. foetidus var. acidus). The rest of the 47 isolates were also identified as A. acidus from their metabolite profile. Neither production of ochratoxin A nor fumonisins B(2) and B(4) by any of the 47 isolates were observed.

Occurrence of fumonisins B(2) and B(4) in retail raisins.

Concerns that raisins may be contaminated by fumonisins stem from the persistent occurrence of Aspergillus niger spores on raisins and the recent discovery of fumonisin production by A. niger on grapes, which leads to the widespread occurrence of fumonisin B(2) in wine. This study presents an LC-MS/MS survey of fumonisins in retail raisins. In 10 of 21 brands collected in Denmark, Germany, and The Netherlands, fumonisins B(2) and B(4) were detected at levels up to 13 and 1.3 μg/kg, respectively. Only fumonisin B(2) has been detected in wine, so the presence of fumonisin B(4) in raisins suggests that the fumonisins are produced mainly during the drying process concomitant with the decreasing water activity. Analysis of multiple packages from one manufacturer showed a 3-fold package-to-package variation, suggesting that a few raisins per package are contaminated.

Single-kernel analysis of fumonisins and other fungal metabolites in maize from South African subsistence farmers

Fumonisins are important Fusarium mycotoxins mainly found in maize and derived products. This study analysed maize from five subsistence farmers in the former Transkei region of South Africa. Farmers had sorted kernels into good and mouldy quality. A total of 400 kernels from 10 batches were analysed; of these 100 were visually characterised as uninfected and 300 as infected. Of the 400 kernels, 15% were contaminated with 1.84–1428 mg kg−1 fumonisins, and 4% (n = 15) had a fumonisin content above 100 mg kg−1. None of the visually uninfected maize had detectable amounts of fumonisins. The total fumonisin concentration was 0.28–1.1 mg kg−1 for good-quality batches and 0.03–6.2 mg kg−1 for mouldy-quality batches. The high fumonisin content in the batches was apparently caused by a small number (4%) of highly contaminated kernels, and removal of these reduced the average fumonisin content by 71%. Of the 400 kernels, 80 were screened for 186 microbial metabolites by liquid chromatography-tandem mass spectrometry, detecting 17 other fungal metabolites, including fusaric acid, equisetin, fusaproliferin, beauvericin, cyclosporins, agroclavine, chanoclavine, rugulosin and emodin. Fusaric acid in samples without fumonisins indicated the possibility of using non-toxinogenic Fusaria as biocontrol agents to reduce fumonisin exposure, as done for Aspergillus flavus. This is the first report of mycotoxin profiling in single naturally infected maize kernels.

Production of fumonisins B2 and B4 in Tolypocladium species

Tolypocladium inflatum is known primarily for its production of the cyclosporines that are used as an immunosuppressive drug. However, we report here the production of the carcinogenic fumonisins B2 and B4 by this biotechnologically relevant fungal genus. These mycotoxins were detected in 11 strains tested from three species: Tolypocladium inflatum, T. cylindrosporum, and T. geodes. Production of fumonisins by Fusarium spp. and Aspergillus niger is highly medium- and temperature-dependent, so the effect of these parameters on fumonisin production by three T. inflatum strains was studied. Maximum production was achieved on media with high sugar content incubated at 25–30°C. Since these results demonstrate that fumonisin production could be widespread within the genus Tolypocladium, the potential contamination of commercial cyclosporine preparations with fumonisins needs to be investigated.

Occurrence of Fumonisins B2 and B4 in Retail Raisins

Concerns that raisins may be contaminated by fumonisins stem from the persistent occurrence of Aspergillus niger spores on raisins and the recent discovery of fumonisin production by A. niger on grapes, which leads to the widespread occurrence of fumonisin B(2) in wine. This study presents an LC-MS/MS survey of fumonisins in retail raisins. In 10 of 21 brands collected in Denmark, Germany, and The Netherlands, fumonisins B(2) and B(4) were detected at levels up to 13 and 1.3 μg/kg, respectively. Only fumonisin B(2) has been detected in wine, so the presence of fumonisin B(4) in raisins suggests that the fumonisins are produced mainly during the drying process concomitant with the decreasing water activity. Analysis of multiple packages from one manufacturer showed a 3-fold package-to-package variation, suggesting that a few raisins per package are contaminated.

Potato carrot agar with manganese as an isolation medium for Alternaria, Epicoccum and Phoma

A semi-selective medium for isolation of Alternaria spp., Epicoccum sp. and Phoma spp. from soil and plant samples was developed. The basal medium was a modified potato carrot agar (PCA), containing 10 g/L of potato and carrot. It is known that the target genera sporulate well on standard PCA when grown at 25 degrees C with an alternating light/dark cycle consisting of 8 h of cool-white daylight followed by 16 h darkness. Addition of 1.5% MnCl(2) 4 H(2)O (w/v) inhibited most other fungi than Alternaria, Epicoccum and Phoma species when tested on pure cultures. The mycobiota of two soil samples and eight grain samples were examined using PCA-Mn and three commonly used isolation media, DRYES, DG18 and V8. On the three conventional media growth of several genera was observed with the predominant being Aspergillus, Eurotium, Fusarium, Mucor, Penicillium and Rhizopus. Of these only F. oxysporum and F. verticillioides were able to grow on PCA-Mn. Alternaria infectoria and Epicoccum nigrum were present in three cereal grain samples, but emerged to a far lower degree on the three conventional media compared to PCA-Mn. Three black spored fungi, identified as Phoma eupyrena, Paraconiothyrium minitan and one unknown species, were isolated from the two soil samples when incubated on PCA-Mn but were absent on the three conventional media.