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Production of toxic metabolites in Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei: justification of mycotoxin testing in food grade enzyme preparations derived from the three fungi.

Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei are three important production organisms used in industrial fermentations. Several of the fungal secondary metabolites produced by selected strains of these three fungi are capable of eliciting toxicity in animals. Among those toxic substances are the well-known mycotoxins 3-nitropropionic acid and ochratoxin A. However, many others, such as kojic acid, may not be true mycotoxins. The production, extraction, chemical structure, and the toxicity (expressed as LD(50)) of these substances are reviewed. Production of toxic secondary metabolites in A. niger, A. oryzae, and T. reesei is strain-specific and environment-dependent. Considering all of the safety measures taken in the industrial production process, these three fungal species are safe to use. The recently revised JECFA specification for mycotoxins in food enzyme preparations is also discussed. The extent of mycotoxin tests in food enzyme preparations should be judged on a case-by-case basis, through a careful evaluation based on knowledge of taxonomy, biochemistry, and genetics. In many cases, the testing scope at the level of genus should be sufficient. In other cases, the scope can even be further narrowed based on scientific knowledge and assessment.

Animals↗

The Cu,Zn superoxide dismutases of Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus: purification and biochemical comparison with the Aspergillus fumigatus Cu,Zn superoxide dismutase.

Cu,Zn superoxide dismutases (SODs) have been purified to homogeneity from Aspergillus flavus and A. niger, which are significant causative agents of aspergillosis, and from A. nidulans and A. terreus, which are much rarer causative agents of disease, using a combination of isoelectric focusing and gel filtration fast protein liquid chromatography. The purified enzymes have been compared with the previously described SOD from the most important pathogen in the genus, A. fumigatus (M. D. Holdom, R. J. Hay, and A. J. Hamilton, Free Radical Res. 22:519-531, 1995). The N-terminal amino acid sequences of the four newly purified enzymes were almost identical and demonstrated homology to known Cu,Zn SODs from a range of organisms including that from the previously described SOD from A. fumigatus. SOD activity was detectable in the culture filtrates of all species, and intracellular Cu,Zn SOD activity as a proportion of total protein was highest in early-log-phase cultures. The specific activities of the purified enzymes were similar, and all four of the newly described enzymes were inhibited by potassium cyanide and diethyldithiocarbamate, known Cu,Zn SOD inhibitors. Sodium azide and o-phenanthroline demonstrated inhibition at concentrations from 5 to 30 mM, and EDTA also exhibited a varying degree of inhibition of SOD activity. However, there were differences in the nonreduced molecular masses, the reduced molecular masses, and the isoelectric points of the four newly described SODs and the A. fumigatus enzyme; these varied from 55 to 123 kDa, 17.5 to 19.5 kDa, and 5.0 to 5.9, respectively. Of particular note was the observation that the A. fumigatus enzyme was thermostable compared with the SODs from the other species; in addition, the A.fiumigatus enzyme retained all of its activity at 37 degrees C relative to 20 degrees C, whereas the SODs of A. nidulans and A. terreus lost significant activity at the higher temperature. Aspergillus Cu,Zn SOD plays a hypothetical role in the avoidance of oxidative killing mechanisms, and our data suggest that the thermotolerant A. fumigatus Cu,Zn SOD would be more effective in such a protective system than, for example, the equivalent enzyme from the more rarely pathogenic A. nidulans.

Amino Acid Sequence↗

In vitro efficacy of 75 essential oils against Aspergillus niger.

Aspergillus niger is an opportunistic human pathogen and a strong air pollutant. A study was conducted with 75 different essential oils for the inhibition of hyphal growth and spore formation in Aspergillus niger. Cinnamomum zeylanicum (bark), Cinnamomum zeylanicum (leaf), Cinnamomum cassia, Syzygium aromaticum and Cymbopogon citratus were the top five essential oils which demonstrated marked inhibitory effect against hyphal growth and spore formation of A. niger. The chemical composition of these five most active essential oils was investigated by gas chromatography-mass spectra (GC-MS). Most of the other essential oils were found challenging to combat A. niger, suggesting their use as strong aroma therapeutic agents.

Aspergillus niger↗

Optimisation of fermentation conditions for gluconic acid production by a mutant of Aspergillus niger.

Aspergillus niger ORS-4, isolated from the sugarcane industry waste materials was found to produce notable level of gluconic acid. From this strain, a mutant Aspergillus niger ORS-4.410 having remarkable increase in gluconic acid production was isolated and compared for fermentation properties. Among the various substrates used, glucose resulted into maximum production of gluconic acid (78.04 g/L). 12% concentration led to maximum production. Effect of spore age and inoculum level on fermentation indicated an inoculum level of 2% of the 4-7 days old spores were best suited for gluconic acid production. Maximum gluconate production could be achieved after 10-12 days of the fermentation at 30 degrees C and at a pH of 5.5. Kinetic analysis of production indicated that growth of the mutant was favoured during initial stages of the fermentation (4-8 days) and production increased during the subsequent 8-12 days of the fermentation. CaCO3 and varying concentrations of different nutrients affected the production of gluconic acid. Analysis of variance for the factors evaluated the significant difference in the production levels.

Aspergillus niger↗

Bioenergetic consequences of glucoamylase production in carbon-limited chemostat cultures of Aspergillus niger.

Aspergillus niger has been grown in glucose- and maltose-limited continuous cultures to determine the bioenergetic consequences of the production of the extracellular enzyme glucoamylase. Growth yields (g biomass per mol substrate) were high, indicating that growth was very efficient and protein production for biomass was not exceedingly energy consuming. It has been found that the energy costs for the production of this extracellular enzyme is very high. Depending on the efficiency of energy conservation the glucoamylase protein yield on ATP is between 1.3 and 2.6 g protein per mol ATP, which is equal or less than 10% of the theoretical maximum of 25.5. These high energy costs most probably have to be invested in the process of excretion. A comparison between an industrial over-producing strain and the wild type Aspergillus niger showed that this over-producing strain most probably is a regulatory mutant. Two regions of specific growth rates could be determined (one at specific growth rates lower and one at specific growth rates higher than 0.1 h-1), which are characterized by differences in mycelium morphology and a significant deviation from linearity in the linear equation for substrate utilization. Analysis of the region of specific growth rates higher than 0.1 h-1 yielded maintenance requirements of virtual zero. It has been concluded that for a good analysis of the growth behaviour of filamentour fungi the linear equation for substrate utilization is not suitable, since it contains no term for the process of differentiation.

Adenosine Triphosphate↗

Circular dichroism studies of acid proteinases from Aspergillus niger and Aspergillus awamori.

Acid proteinases produced by strains of Aspergillus niger and Aspergillus awamori were isolated by means of ethanol precipitation, gel filtration and anion-exchange high resolution chromatography. In each case, the purified proteinase showed a single band in polyacrylamide gel electrophoresis. Their molecular weights were almost identical (approx. 45,000). However, the proteinase from Aspergillus awamori contained 16% of neutral hexoses while the other enzyme (Aspergillus niger) showed negligible amounts of these carbohydrates. Both enzymes displayed circular dichroism spectra that share a number of features with that of penicillopepsin. This suggests that proteinases from Aspergilli possess the structural folding pattern typical of aspartic proteinases. Proteolytic-activity pH optima were different, thus distinguishing one enzyme from another. This variation seems to be related to the particular resistance of the proteinases to acid denaturation, as indicated by changes in their circular dichroism spectra when the pH is decreased.

Aspartic Acid Endopeptidases↗

Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger.

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.

Aflatoxin B1↗

Glucoamylase of Aspergillus niger.

Aspergillus niger produces two extracellular glucoamylases (GAI of Mr 85 300 and GAII of Mr 77 600) separable on DEAE-cellulose. The enzymes differes in electrophoretic mobility, thermostability and substrate specificity. The GAI/GAII ratio depends on the concentration and form of nitrogen (nitrate or ammonium) in the culture medium. Proteinase VIII from Bacillus subtilis converts GAI to a form showing properties similar to those of GAII. Possible proteolytic degradation of GAI to GAII by Asp. niger endogenous proteinase(s) is suggested.

Aspergillus niger↗

Metabolism of dimethylterephthalate by Aspergillus niger.

Aspergillus niger (AG-1) metabolized dimethylterephthalate through monomethylterephthalate, terephthalate and protocatechuate. Degradation of dimethylterephthalate was followed by extraction of residual dimethylterephthalate from the spent medium. The quantitative UV analysis showed that 58% of the dimethylterephthalate supplement was taken up in 144 h. The metabolites were isolated from resting cell cultures. Thin layer chromatography analysis of the extract revealed the presence of two intermediates, monomethylterephthalate and terephthalate. Use of an inhibitor in resting cell culture experiment demonstrated the accumulation of protocatechuate. The time course of protocatechuate accumulation was also studied. Metabolites were identified by employing various physicochemical methods. Enzyme studies using cell-free extracts exhibited dimethylterephthalate esterase and protocatechuate dioxygenase activities. Protocatechuate was oxidized by the meta cleavage pathway. A tentative pathway for the degradation of DMTP has been proposed in A. niger.

Aspergillus niger↗

Release of ferulic acid from sugar-beet pulp by using arabinanase, arabinofuranosidase and an esterase from Aspergillus niger.

Aspergillus niger cinnamoyl esterase (CinnAE) is shown to be active towards a wide range of feruloylated oligosaccharides derived from sugar-beet pulp (SBP). The esterase hydrolysed ferulic acid ester-linked to either C-2 of arabinose or C-6 of galactose residues, and demonstrated the highest activity towards the feruloylated arabinose trisaccharide. However, CinnAE was able to release only 0.88% of total alkali-extractable ferulic acid from SBP in 24 h when acting alone. To determine whether cell-wall-degrading enzymes could increase the release of ferulic acid by CinnAE, SBP was incubated with various carbohydrases [cellulase, polygalacturonase, endo-arabinanase, alpha-L-arabinofuranosidase, endo-(1,4-beta-D-galactanase, beta-D-galactosidase]. These were added alone and in pairs, both in the presence and absence of CinnAE. We showed that all the carbohydrases tested were free of esterase activity. When individual carbohydrases were incubated with SBP, whether in the presence or absence of CinnAE, less than 1% of the feruloyl groups were released. When incubated with a mixture of endo-arabinanase and alpha-L-arabinofuranosidase, the esterase was able to release 14 times more of the alkali-extractable ferulic acid present in the whole pulp as free acid than CinnAE alone. Ferulic acid is linked either to L-arabinose or D-galactose in SBP, but no corresponding increase in ferulic acid release was detected when SBP was incubated with CinnAE plus endo-(1,4)-beta-D-galactanase and beta-D-galactosidase (both from A. niger). Hence feruloylated arabinans in SBP are readily available for hydrolysis by arabinan-degrading enzymes, whereas feruloylated galactans are not available for hydrolysis by galactan-degrading enzymes.

Aspergillus niger↗

Dye biosorption sites in Aspergillus niger.

Aspergillus niger is capable of removing dyes from an aqueous solution. In the study, the roles played by three major functional groups: carboxyl, amino and phosphate, and the lipid fraction in the biomass of A. niger in biosorption of four dyes, Basic Blue 9, Acid Blue 29, Congo Red and Disperse Red 1, were investigated. These functional groups in A. niger were chemically modified individually to determine their contribution to the biosorption of dyes. It was found that biosorption of dyes was influenced by the functional groups in the fungal biomass and the chemical structure of the dyes.

Absorption↗

Chronic bilateral otomycosis caused by Aspergillus niger.

Aspergillus niger, an opportunistic filamentous fungus, was identified as the cause of chronic bilateral otomycosis in a 46-year-old female patient who was unresponsive to different drugs. The patient showed signs of erythema, otalgia, itching, otorrhoea and presence of greyish black coloured mass in both the ear canals. The direct microscopical examination of the ear debris in potassium hydroxide preparations, Giemsa, phase contrast and Gram revealed many thin, branched septate hyphae, condia and conidiophores morphologically indistinguishable from Aspergillus spp. The histopathological section of the ear wax mass by haematoxylin and eosin and periodic acid-Schiff techniques also showed similar fungal elements. The patient responded to 1% solution of mercurochrome. The use of mercurochrome in developing countries like India may be recommended to treat the fungal otitis in patients. We also emphasize that 'Narayan' stain should be routinely employed by microbiology and public health laboratories to study the morphology of pathogenic fungi.

Anti-Infective Agents, Local↗

Arginine and proline genes of Aspergillus niger.

Aspergillus niger mutants defective in arginine or proline biosynthesis have been isolated and 12 genetic loci were identified. Mutation was induced by low doses UV, and mutants were isolated after filtration enrichment. The mutants were classified according to their phenotype in growth tests and were further characterized in complementation tests. The arginine auxotrophic mutants represent nine complementation groups. Three additional complementation groups were found for mutants that could grow on proline (two of them on arginine too). Linkage group analysis was done in somatic diploids obtained from a mutant and a master strain with genetic markers on six chromosomes. The arg genes belong to six different linkage groups and the pro genes to two. One arg-mutant could be complemented by transformation with the A. nidulans argB+ gene, and this A. niger gene thus appeared to be homologous to the A. nidulans argB. We isolated an A. niger strain with the argB gene tightly linked with the nicA1 marker. This strain is very suitable as acceptor for transformation with an argB-plasmid, because transformants with inserts on the homologous site can be recognized and analyzed genetically using the nicA1 marker gene.

Arginine↗

Utilization of brewery spent grain liquor by Aspergillus niger.

Aspergillus niger was found capable of rapidly converting about 97% of the sugar from brewery spent grain liquor to fungal mass. The yield of dry mycelium, based on the sugar consumed, was approximately 57%. This fungus produced 1.10% titratable acid calculated as citric acid and reduced the biochemical oxygen demand by 96%.

Aspergillus↗

Metabolism of polycyclic aromatic hydrocarbons by Aspergillus niger.

Aspergillus niger metabolised polycyclic aromatic hydrocarbons such as naphthalene, anthracene and phenanthrene. The maximum weight of mycelium was obtained at 8-10 days of incubation. TLC and HPLC analysis revealed the accumulation of metabolites in resting cell culture. The metabolism of naphthalene and anthracene follows the gentisate pathway, where as phenenthrene was metabolised via protocatechuate in this organism.

Aspergillus niger↗

Novel metabolites in phenanthrene and pyrene transformation by Aspergillus niger.

Aspergillus niger, isolated from hydrocarbon-contaminated soil, was examined for its potential to degrade phenanthrene and pyrene. Two novel metabolites, 1-methoxyphenanthrene and 1-methoxypyrene, were identified by conventional chemical techniques. Minor metabolites identified were 1- and 2-phenanthrol and 1-pyrenol. No 14CO2 evolution was observed in either [14C]phenanthrene or [14C]pyrene cultures.

Alkaloids↗

Degradation of orcinol by Aspergillus niger.

Aspergillus niger could utilize orcinol (5-methyl-resorcinol or 3,5-dihydroxytoluene) as the sole source of carbon and energy. In the first step of catabolism A. niger hydroxylates orcinol to form 2,3,5-trihydroxytoluene. Its oxidized form, 2-hydroxy-6-methyl-1,4-benzoquinone, was also formed in the culture medium during growth of this organism. Orcinol-grown cells showed a net increase in the intracellular acetate pool, compared with glucose-grown cells. Cell-free extracts of orcinol-grown cells showed higher activity of orcinol hydroxylase, catechol 1,2-oxygenase, and isocitrate lyase than that of glucose-grown cells. Both orcinol-grown and resorcinol-grown cells exhibit similar respiratory activity on all the substrates checked.

Aspergillus niger↗